Allogeneic cell therapy of b cell malignancies using genetically engineered t cells targeting cd19

ABSTRACT

A population of genetically engineered immune cells (e.g., T cells), which express a chimeric antigen receptor (CAR) specific to CD19 and contain a disrupted TRAC gene, a disrupted B2M gene, or both, for use in treating a B cell malignancy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing dates of U.S.Provisional Application No. 62/840,913, filed Apr. 30, 2019, the entirecontents of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Chimeric antigen receptor (CAR) T cell therapies are adoptive T celltherapeutics used to treat human malignancies. Although CAR T celltherapy has led to tremendous clinical success, including durableremission in relapsed/refractory non-Hodgkin lymphoma (NHL) andpediatric acute lymphoblastic leukemia (ALL), the approved products areautologous and require patient-specific cell collection andmanufacturing. Because of this, some patients have experienced diseaseprogression or death while awaiting treatment. Accordingly, thereremains a need for improved CAR T cell therapeutics.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the development ofallogeneic cell therapy for B cell malignancies such as transformed FLor DLBCL using genetically engineered T cells (e.g., CTX110 cells,a.k.a., TC1 cells) expressing an anti-CD19 chimeric antigen receptor(CAR) and having disrupted TRAC gene and B2M gene. The allogeneic CAR-Tcell therapy disclosed herein showed treatment efficacies in humanpatients having B cell malignancies disclosed herein, including completeresponses in certain patients and long durability of responses. Further,the allogeneic CAR-T cell therapy disclosed herein exhibited desiredpharmacokinetic features in the human patients, including prolongedCAR-T cell expansion and persistence after infusion.

Accordingly, some aspects of the present disclosure provides a methodfor treating a B-cell malignancy in a human patient, the methodcomprising: (i) subjecting a human patient having a B-cell malignancy toa lymphodepletion treatment; and (ii) administering to the human patienta population of genetically engineered T cells after step (i). In someembodiments, step (i) can be performed about 2-7 days prior to step(ii). In some embodiments, the population of genetically engineered Tcells is allogeneic.

The population of the genetically engineered T cells may comprise Tcells that comprise: (a) a disrupted T cell receptor alpha constant(TRAC) gene, (b) a nucleic acid coding for a chimeric antigen receptor(CAR) that binds CD19, wherein the CAR comprises an anti-CD19 singlechain variable fragment (scFv) that comprises a heavy chain variableregion set forth in SEQ ID NO: 51, and a light chain variable region setforth in SEQ ID NO: 52, and wherein the nucleic acid is inserted in thedisrupted TRAC gene, and (c) a disrupted beta 2-microglobulin (β2M)gene. In some embodiments, the disrupted TRAC gene comprises a deletionof a fragment comprising the nucleotide sequence of SEQ ID NO: 26.

In some embodiments, the population of genetically engineered T cells isadministered to the human patient at a dose of about 1×10⁷ to about1×10⁹ CAR⁺ T cells. In some examples, the population of geneticallyengineered T cells is administered to the human patient at a dose ofabout 1×10⁷ CAR⁺ T cells. In some examples, the population ofgenetically engineered T cells is administered to the human patient at adose of about 3×10⁷ CAR T cells. In some examples, the population ofgenetically engineered T cells is administered to the human patient at adose of about 1×10⁸ CAR⁺ T cells. In some examples, the population ofgenetically engineered T cells is administered to the human patient at adose of about 3×10⁸ CAR⁺ T cells. In some examples, the population ofgenetically engineered T cells is administered to the human patient at adose of about 1×10⁹ CAR⁺ T cells. In any event, the population ofgenetically engineered T cells administered to the human patient perdose contains no more than 7×10⁴ TCR⁺ T cells/kg.

In some embodiments, the lymphodepletion treatment in step (i) comprisesco-administration to the human patient fludarabine at about 30 mg/m² andcyclophosphamide at about 500-750 mg/m² per day for three days. Forexample, the lymphodepletion treatment in step (i) comprisesco-administration to the human patient fludarabine at about 30 mg/m² andcyclophosphamide at about 500 mg/m² per day for three days. In otherexamples, the lymphodepletion treatment in step (i) comprisesco-administration to the human patient fludarabine at about 30 mg/m² andcyclophosphamide at about 750 mg/m² per day for three days.

In some embodiments, prior to step (i), the human patient does not showone or more of the following features: (a) significant worsening ofclinical status, (b) requirement for supplemental oxygen to maintain asaturation level of greater than 91%, (c) uncontrolled cardiacarrhythmia, (d) hypotension requiring vasopressor support, (e) activeinfection, and (f) grade ≥2 acute neurological toxicity.

In some embodiments, after step (i) and prior to step (ii), the humanpatient does not show one or more of the following features: (a) activeuncontrolled infection; (b) worsening of clinical status compared to theclinical status prior to step (i); and (c) grade ≥2 acute neurologicaltoxicity.

Any of the methods disclosed herein may further comprise (iii)monitoring the human patient for development of acute toxicity afterstep (ii); and (iv) managing the acute toxicity if occurs. In someembodiments, step (iii) can be performed for at least 28 days afteradministration of the population of genetically engineered T cells.Exemplary acute toxicity may comprise tumor lysis syndrome (TLS),cytokine release syndrome (CRS), immune effector cell-associatedneurotoxicity syndrome (ICANS), B cell aplasia, hemophagocyticlymphohistiocytosis (HLH), cytopenia, graft-versus-host disease (GvHD),hypertension, renal insufficiency, or a combination thereof.

In some embodiments, the B cell malignancy is non-Hodgkin lymphoma.Examples include, but are not limited to, diffuse large B cell lymphoma(DLBCL), high grade B cell lymphoma with MYC and BCL2 and/or BCL6rearrangement, transformed follicular lymphoma (FL), or grade 3b FL. Insome instances, DLBCL is DLBCL not otherwise specified (NOS). In someexamples, the B cell malignancy is refractory and/or relapsed.

In some embodiments, the human patient may have at least one measurablelesion that is fluorodeoxyglucose positron emission tomography(PET)-positive. In some embodiments, the human patient has undergone oneor more lines of prior anti-cancer therapies. In some examples, thehuman patient has undergone two or more lines of prior anti-cancertherapies. Exemplary prior anti-cancer therapies may comprise ananti-CD20 antibody, an anthracycline-containing regimen, or acombination thereof.

In some examples, the human patient has refractory or relapsedtransformed FL and has undergone at least one line of chemotherapy fordisease after transformation to DLBCL. In other examples, the B cellmalignancy is refractory, and the human patient has progressive diseaseon last therapy, or has stable disease following at least two cycles oftherapy with duration of stable disease of up to 6 months. In yet otherexamples, the human patient has failed prior autologous hematopoieticstem cell transplantation (HSCT) or ineligible for prior autologousHSCT. Alternatively or in addition, the human patient is subject to anadditional anti-cancer therapy after treatment with the population ofgenetically engineered T cells.

In any of the methods disclosed herein, the human patient has one ormore of the following features:

(a) has an Eastern Cooperative Oncology Group (ECOG) performance status0 or 1;

(b) adequate renal, liver, cardiac, and/or pulmonary function;

(c) free of prior gene therapy or modified cell therapy;

(d) free of prior treatment comprising an anti-CD19 antibody;

(e) free of prior allogeneic HSCT;

(f) free of detectable malignant cells from cerebrospinal fluid;

(g) free of brain metastases;

(h) free of prior central nervous system disorders;

(i) free of unstable angina, arrhythmia, and/or myocardial infarction;

(j) free of uncontrolled infection;

(k) free of immunodeficiency disorders or autoimmune disorders thatrequire immunosuppressive therapy; and

(l) free of infection by human immunodeficiency virus, hepatitis Bvirus, or hepatitis C virus.

In any of the methods disclosed herein, the anti-CD19 CAR expressed bythe genetically engineered T cells may comprise an extracellular antigenbinding domain, which is an anti-CD19 scFv comprising the amino acidsequence of SEQ ID NO: 47. In some embodiments, the anti-CD19 CAR maycomprise the amino acid sequence of SEQ ID NO: 40.

In some embodiments, the nucleic acid encoding the anti-CD19 CAR isinserted at the site of deletion in the disrupted TRAC gene. In someexamples, the disrupted TRAC gene comprises the nucleotide sequence ofSEQ ID NO: 54. Alternatively or in addition, the disrupted β2M gene inthe population of genetically engineered T cells comprises at least oneof the nucleotide sequence set forth in SEQ ID NOs: 9-14.

In some embodiments, at least 90% of the T cells in the population ofgenetically engineered T cells do not express a detectable level of TCRsurface protein. For example, at least 70% of the T cells in thepopulation of genetically engineered T cells do not express a detectablelevel of TCR surface protein; at least 50% of the T cells in thepopulation of genetically engineered T cells do not express a detectablelevel of B2M surface protein; and/or at least 30% of the T cells in thepopulation of genetically engineered T cells express a detectable levelof the CAR. In some examples, at least 99.5% of the T cells in thepopulation of genetically engineered T cells do not express a detectablelevel of TCR surface protein. In some examples, at least 70% of the Tcells in the population of genetically engineered T cells do not expressa detectable level of B2M surface protein. In specific examples, atleast 85% of the T cells in the population of the genetically engineeredT cells do not express a detectable level of B2M surface protein. Insome examples, at least 50% of the T cells in the population ofgenetically engineered T cells express a detectable level of the CAR. Inspecific examples, at least 70% of the T cells in the population ofgenetically engineered T cells express a detectable level of the CAR.

In a specific example, the population of genetically engineered T cellsfor use in any of the methods disclosed herein are CTX110 cells.

In any of the methods disclosed herein, the population of geneticallyengineered T cells are administered to the human patient via intravenousinfusion. In some examples, the population of genetically engineered Tcells may be suspended in a cryopreservation solution.

Also within the scope of the present disclosure are pharmaceuticalcompositions for use in treating a B-cell malignancy, the pharmaceuticalcomposition comprising any of the population of genetically engineered Tcells disclosed herein (e.g., the CTX110 cells), as well as use of thegenetically engineered T cells for manufacturing a medicament for use intreating a B-cell malignancy as disclosed herein.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of flow cytometry plots of human primary T-cells,TRAC−/B2M−CD19CAR+T cells (TC1), 8 days post-editing. The graphs showreduced surface expression of TRAC and B2M. TCR/MHC I double knockoutcells express high levels of the CAR transgene (bottom panel). Negativeselection of TC1 cells with purification beads leads to a reduction inTCR positive cells (right panel).

FIG. 2 is a graph depicting high editing rates achieved at the TRAC andB2M loci in TRAC−/B2M−CD19CAR+T cells (TC1). Surface expression of TCRand MHCI, which is the functional output of gene editing, was measuredand plotted as editing percentage on the y-axis. High efficiency (e.g.,greater than 50%) site-specific integration and expression of the CARfrom the TRAC locus were detected. These data demonstrate greater than50% efficiency for the generation of TRAC−/B2M−/anti-CD19CAR+ T cells.

FIG. 3 is a graph depicting a statistically significant decrease intumor volume (mm³) (p=0.007) in NOG Raji mice following treatment withTRAC−/β2M−/CD19 CAR+ T cells (TC1).

FIG. 4 is a survival curve graph demonstrating increased survival of NOGRaji mice treated with TC1 cells in comparison to NOG Raji micereceiving no treatment.

FIG. 5 is a survival curve graph demonstrating increased survival of NOGRaji mice treated with TC1 cells on day 4, in comparison to control micereceiving no treatment on day 1.

FIGS. 6A and 6B include diagrams showing persistence and anti-tumoractivity of TC1 cells in mice. 6A: a series of flow cytometry plotsdemonstrating that TC1 cells persist in NOG Raji mice. 6B: a graphdemonstrating that TC1 cells selectively eradicate splenic Raji cells inNOG Raji mice treated with TC1 in comparison to controls (NOG Raji micewith no treatment or NOG mice). The effect is depicted as a decreasedsplenic mass in NOG Raji mice treated with TC1 in comparison tocontrols.

FIG. 7 is a series of flow cytometry plots demonstrating that persistentsplenic TC1 cells are edited in two independent NOG Raji mice with TC1treatment.

FIG. 8 is a Kaplan-Meier survival plot demonstrating increased survivalof NOG Nalm6 mice treated with TC1 cells on day 4, in comparison tocontrol mice receiving no treatment on day 1.

FIG. 9 is a Kaplan-Meier survival plot demonstrating an increasesurvival of mice bearing a disseminated Nalm6 B-cell acute lymphoblasticleukemia (B-ALL) after treatment with different concentrations of TC1,in comparison to control mice receiving no treatment.

FIG. 10 is a graph depicting a statistically significant inhibition intumor cell expansion in the disseminated Nalm6 B-cell acutelymphoblastic leukemia (B-ALL) tumor model following treatment with TC1cells.

FIG. 11 is a Kaplan-Meier survival plot of healthy mice treated with TC1cells or various control cells (PBMCs or electroporated (EP) T cells)after radiation, or mice that only received radiation (“RT only”).

FIG. 12 is a graph showing percentage of body weight change of the micetreated in FIG. 18.

FIG. 13 is a Kaplan-Meier survival plot of healthy mice treated with alow dose (2×10⁷) or high dose (4×10⁷) of TC1 cells, or unedited T cellsafter radiation, or mice that only received radiation (“Vehicle-RT”).

FIG. 14 is a graph showing percentage of body weight change of the micetreated in FIG. 20, in addition to mice that were not irradiated and notdosed with cells (“Vehicle—no RT”).

FIG. 15 is a bar graph showing percentage of CD27+CD45RO− cells withinthe unedited CD8+ T cell subset of peripheral blood cells from sixdifferent donors.

FIG. 16 provides flow cytometry results of TCRαβ and B2M expression onTC1 cells before and after depletion of TCRαβ+ cells.

FIG. 17 is a graph the percentage loss of protein for TCR− and MHCI-(B2M) after gene editing, and percentage of cells expressing ananti-CD19 CAR in edited TC1 cells from individual lots of TC1production.

FIG. 18 provides graphs showing the percentage of PD1+(top left),LAG3+(top right), TIM3+(bottom left) or CD57+(bottom right) in the Tcell population from six different donors before and after editing.

FIG. 19 is a graph showing the percentage of cell lysis of CD19-positivecell lines (Nalm6; Raji; and K562-CD19) and CD19-negative cells (K562)when co-cultured at different ratios with TC1 cells or unedited T cells.

FIG. 20 is a graph showing the number of viable TC1 cells when culturedin the presence of T-cell media (serum+IL2+IL7; Complete Media), mediacontaining serum but no IL2 or IL7 cytokines (5% Serum, No cytokines) orno serum or cytokines (No Serum, No Cytokines). Cells were counted onthe indicated days post gene editing. Mean values from three lotsshown±SD.

FIG. 21 is a schematic depicting the clinical study design to evaluateCTX110 cells, (a.k.a., TC1 cells) administered after lymphodepletion tohuman subjects having CD19+ malignancies.

DETAILED DESCRIPTION OF THE INVENTION

Cluster of Differentiation 19 (CD19) is an antigenic determinantdetectable on leukemia precursor cells. The human and murine amino acidand nucleic acid sequences can be found in a public database, such asGenBank, UniProt and Swiss-Prot. For example, the amino acid sequence ofhuman CD19 can be found as UniProt/Swiss-Prot Accession No. P15391 andthe nucleotide sequence encoding of the human CD19 can be found atAccession No. NM_001178098. CD19 is expressed on most B lineage cancers,including, e.g., acute lymphoblastic leukemia, chronic lymphocyteleukemia and non-Hodgkin's lymphoma. It is also an early marker of Bcell progenitors. See, e.g., Nicholson et al. Mol. Immun. 34 (16-17):1157-1165 (1997).

The present disclosure provides an allogeneic CAR-T cell therapy for Bcell malignancies. The CAR-T cell therapy involves a population ofgenetically engineered T cells expressing an anti-CD19 CAR and havingdisrupted TRAC gene and B2M gene, the nucleic acid coding for theanti-CD19 CAR being inserted into the TRAC gene locus, therebydisrupting expression of the TRAC gene. The allogenic anti-CD19 CAR-Tcells are prepared using parent T cells obtained from healthy donors. Assuch, the CAR-T therapy is available to a patient having the target Bcell malignancy immediately after diagnosis, as opposed to at leastthree week gap between diagnosis and treatment in autologous CAR-Ttherapy required for manufacturing the CAR-T cells from the patient'sown T cells. The allogeneic CAR T therapy can be stored and inventoriedat the site of care to facilitate treatment immediately followingdiagnosis. The immediate availability of the allogeneic anti-CD19 CAR Ttherapy eliminates the need for bridging chemo-therapy, which may berequired when autologous CAR-T cells are manufactured from the patient'sown cells. The allogeneic anti-CD19 CAR-T cell therapy disclosed hereinshowed treatment efficacies in human patients having B cell malignanciesdisclosed herein, including complete responses in certain patients andlong durability of responses. Further, the allogeneic CAR-T cell therapydisclosed herein exhibited desired pharmacokinetic features in the humanpatients, including prolonged CAR-T cell expansion and persistence afterinfusion.

Accordingly, provided herein are methods for treating a B-cellmalignancy in a human patient using a population of geneticallyengineered immune cells such as T cells, which collectively comprises adisrupted TRAC gene, a disrupted B2M, and a nucleic acid encoding ananti-CD19 CAR (e.g., SEQ ID NO: 40, encoded by SEQ ID NO:39). Thenucleic acid encoding the anti-CD19 CAR and optionally comprising apromoter sequence and one or more regulatory elements may be inserted inthe disrupted TRAC gene locus, e.g., replacing the segment of SEQ ID NO:26 in the TRAC gene. The human patient is subject to a lymphodepletiontreatment prior to administration of the population of geneticallyengineered T cells.

I. Anti-CD19 CAR T Cells

Disclosed herein are anti-CD19 CAR T cells (e.g., CTX110 cells) for usein treating B cell malignancies. In some embodiments, the anti-CD19 CART cells are human T cells expressing an anti-CD19 CAR and having adisrupted TRAC gene, a disrupted B2M gene, or a combination thereof. Inspecific examples, the anti-CD19 CART cells express an anti-CD19 CAR andhave endogenous TRAC and B2M genes disrupted.

(i) Anti-CD19 Chimeric Antigen Receptor (CAR)

The genetically engineered immune cells such as T cells disclosed hereexpress a chimeric antigen receptor (CAR) that binds CD19 (an anti-CD19CAR). A chimeric antigen receptor (CAR) refers to an artificial immunecell receptor that is engineered to recognize and bind to an antigenexpressed by undesired cells, for example, disease cells such as cancercells. A T cell that expresses a CAR polypeptide is referred to as a CART cell. CARs have the ability to redirect T-cell specificity andreactivity toward a selected target in a non-MHC-restricted manner. Thenon-MHC-restricted antigen recognition gives CAR-T cells the ability torecognize an antigen independent of antigen processing, thus bypassing amajor mechanism of tumor escape. Moreover, when expressed on T-cells,CARs advantageously do not dimerize with endogenous T-cell receptor(TCR) alpha and beta chains.

There are various generations of CARs, each of which contains differentcomponents. First generation CARs join an antibody-derived scFv to theCD3zeta ((or z) intracellular signaling domain of the T-cell receptorthrough hinge and transmembrane domains. Second generation CARsincorporate an additional co-stimulatory domain, e.g., CD28, 4-1BB(41BB), or ICOS, to supply a costimulatory signal. Third-generation CARscontain two costimulatory domains (e.g., a combination of CD27, CD28,4-1BB, ICOS, or OX40) fused with the TCR CD3ζ chain. Maude et al.,Blood. 2015; 125(26):4017-4023; Kakarla and Gottschalk, Cancer J. 2014;20(2):151-155). Any of the various generations of CAR constructs iswithin the scope of the present disclosure.

Generally, a CAR is a fusion polypeptide comprising an extracellulardomain that recognizes a target antigen (e.g., a single chain fragment(scFv) of an antibody or other antibody fragment) and an intracellulardomain comprising a signaling domain of the T-cell receptor (TCR)complex (e.g., CD3ζ) and, in most cases, a co-stimulatory domain.(Enblad et al., Human Gene Therapy. 2015; 26(8):498-505). A CARconstruct may further comprise a hinge and transmembrane domain betweenthe extracellular domain and the intracellular domain, as well as asignal peptide at the N-terminus for surface expression. Examples ofsignal peptides include MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 30) andMALPVTALLLPLALLLHAARP (SEQ ID NO: 31). Other signal peptides may beused.

The anti-CD19 CAR may comprise an anti-CD19 single-chain variablefragment (scFv) specific for CD19, followed by hinge domain andtransmembrane domain (e.g., a CD8 hinge and transmembrane domain) thatis fused to an intracellular co-signaling domain (e.g., a CD28co-stimulatory domain) and a CD3ζ signaling domain. Exemplary componentsfor use in constructing the anti-CD19 CAR disclosed herein can be foundin the Sequence Table provided below.

(a) Antigen Binding Extracellular Domain

The antigen-binding extracellular domain is the region of a CARpolypeptide that is exposed to the extracellular fluid when the CAR isexpressed on cell surface. In some instances, a signal peptide may belocated at the N-terminus to facilitate cell surface expression. In someembodiments, the antigen binding domain can be a single-chain variablefragment (scFv, which may include an antibody heavy chain variableregion (V_(H)) and an antibody light chain variable region (V_(L)) (ineither orientation). In some instances, the V_(H) and V_(L) fragment maybe linked via a peptide linker. The linker, in some embodiments,includes hydrophilic residues with stretches of glycine and serine forflexibility as well as stretches of glutamate and lysine for addedsolubility. The scFv fragment retains the antigen-binding specificity ofthe parent antibody, from which the scFv fragment is derived. In someembodiments, the scFv may comprise humanized V_(H) and/or V_(L) domains.In other embodiments, the V_(H) and/or V_(L) domains of the scFv arefully human.

The antigen-binding extracellular domain in the CAR polypeptidedisclosed herein is specific to CD19 (e.g., human CD19). In someexamples, the antigen-binding extracellular domain may comprise a scFvextracellular domain capable of binding to CD19. The anti-CD19 scFv maycomprise a heavy chain variable domain (V_(H)) having the same heavychain complementary determining regions (CDRs) as those in SEQ ID NO: 51and a light chain variable domain (V_(L)) having the same light chainCDRs as those in SEQ ID NO: 52. Two antibodies having the same V_(H)and/or V_(L) CDRs means that their CDRs are identical when determined bythe same approach (e.g., the Kabat approach, the Chothia approach, theAbM approach, the Contact approach, or the IMGT approach as known in theart. See, e.g., bioinf.org.uk/abs/). In some examples, the anti-CD19scFv comprises the V_(H) of SEQ ID NO: 51 and/or the V_(L) of SEQ ID NO:52. In specific examples, the anti-CD19 scFv may comprise the amino acidsequence of SEQ ID NO: 47.

(b) Transmembrane Domain

The anti-CD19 CAR polypeptide disclosed herein may contain atransmembrane domain, which can be a hydrophobic alpha helix that spansthe membrane. As used herein, a “transmembrane domain” refers to anyprotein structure that is thermodynamically stable in a cell membrane,preferably a eukaryotic cell membrane. The transmembrane domain canprovide stability of the CAR containing such.

In some embodiments, the transmembrane domain of a CAR as providedherein can be a CD8 transmembrane domain. In other embodiments, thetransmembrane domain can be a CD28 transmembrane domain. In yet otherembodiments, the transmembrane domain is a chimera of a CD8 and CD28transmembrane domain. Other transmembrane domains may be used asprovided herein. In one specific example, the transmembrane domain inthe anti-CD19 CAR is a CD8a transmembrane domain having the amino acidsequence of SEQ ID NO: 32.

(c) Hinge Domain

In some embodiments, a hinge domain may be located between anextracellular domain (comprising the antigen binding domain) and atransmembrane domain of a CAR, or between a cytoplasmic domain and atransmembrane domain of the CAR. A hinge domain can be any oligopeptideor polypeptide that functions to link the transmembrane domain to theextracellular domain and/or the cytoplasmic domain in the polypeptidechain. A hinge domain may function to provide flexibility to the CAR, ordomains thereof, or to prevent steric hindrance of the CAR, or domainsthereof.

In some embodiments, a hinge domain may comprise up to 300 amino acids(e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In someembodiments, one or more hinge domain(s) may be included in otherregions of a CAR. In some embodiments, the hinge domain may be a CD8hinge domain. Other hinge domains may be used.

(d) Intracellular Signaling Domains

Any of the anti-CD19 CAR constructs disclosed herein contain one or moreintracellular signaling domains (e.g., CD3ζ, and optionally one or moreco-stimulatory domains), which are the functional end of the receptor.Following antigen recognition, receptors cluster and a signal istransmitted to the cell.

CD3ζ is the cytoplasmic signaling domain of the T cell receptor complex.CD3ζ contains three (3) immunoreceptor tyrosine-based activation motif(ITAM)s, which transmit an activation signal to the T cell after the Tcell is engaged with a cognate antigen. In many cases, CD3ζ provides aprimary T cell activation signal but not a fully competent activationsignal, which requires a co-stimulatory signaling. In some examples, theanti-CD19 CAR construct disclosed herein comprise a CD3ζ cytoplasmicsignaling domain, which may have the amino acid sequence of SEQ ID NO:38.

In some embodiments, the anti-CD19 CAR polypeptides disclosed herein mayfurther comprise one or more co-stimulatory signaling domains. Forexample, the co-stimulatory domains of CD28 and/or 4-1BB may be used totransmit a full proliferative/survival signal, together with the primarysignaling mediated by CD3ζ. In some examples, the CAR disclosed hereincomprises a CD28 co-stimulatory molecule, for example, a CD28co-stimulatory signaling domain having the amino acid sequence of SEQ IDNO:36. In other examples, the CAR disclosed herein comprises a 4-1BBco-stimulatory molecule, for example, a 4-1BB co-stimulatory signalingdomain having the amino acid sequence of SEQ ID NO: 34.

In specific examples, an anti-CD19 CAR disclosed herein may include aCD3ζ signaling domain (e.g., SEQ ID NO: 38) and a CD28 co-stimulatorydomain (e.g., SEQ ID NO: 36).

It should be understood that methods described herein encompasses morethan one suitable CAR that can be used to produce genetically engineeredT cells expressing the CAR, for example, those known in the art ordisclosed herein. Examples can be found in, e.g., InternationalApplication Number PCT/IB2018/001619, filed May 11, 2018, whichpublished as WO 2019/097305A2, and International Application NumberPCT/IB2019/000500, filed May 10, 2019, the relevant disclosures of eachof the prior applications are incorporated by reference herein for thepurpose and subject matter referenced herein.

In specific examples, the anti-CD19 CAR disclosed herein may comprisethe amino acid sequence of SEQ ID NO: 40, which may be encoded by thenucleotide sequence of SEQ ID NO: 39. See the sequence table providedbelow.

In the genetically engineered T cells disclosed herein, a nucleic acidcomprising the coding sequence of the anti-CD19 CAR, and optionallyregulatory sequences for expression of the anti-CD19 CAR (e.g., apromoter such as the EF1a promoter provided in the sequence Table) maybe inserted into a genomic locus of interest. In some examples, thenucleic acid is inserted in the endogenous TRAC gene locus, therebydisrupting expression of the TRAC gene. In specific examples, thenucleic acid may replace a fragment in the TRAC gene, for example, afragment comprising the nucleotide sequence of SEQ ID NO: 26.

(ii) Knock-Out of TRAC and B2M Genes

The anti-CD19 CAR-T cells disclosed herein may further have a disruptedTRAC gene, a disrupted B2M gene, or a combination thereof. Thedisruption of the TRAC locus results in loss of expression of the T cellreceptor (TCR) and is intended to reduce the probability of Graft versusHost Disease (GvHD), while the disruption of the β2M locus results inlack of expression of the major histocompatibility complex type I (MHCI) proteins and is intended to improve persistence by reducing theprobability of host rejection. The addition of the anti-CD19 CAR directsthe modified T cells towards CD19-expressing tumor cells.

As used herein, the term “a disrupted gene” refers to a gene containingone or more mutations (e.g., insertion, deletion, or nucleotidesubstitution, etc.) relative to the wild-type counterpart so as tosubstantially reduce or completely eliminate the activity of the encodedgene product. The one or more mutations may be located in a non-codingregion, for example, a promoter region, a regulatory region thatregulates transcription or translation; or an intron region.Alternatively, the one or more mutations may be located in a codingregion (e.g., in an exon). In some instances, the disrupted gene doesnot express or expresses a substantially reduced level of the encodedprotein. In other instances, the disrupted gene expresses the encodedprotein in a mutated form, which is either not functional or hassubstantially reduced activity. In some embodiments, a disrupted gene isa gene that does not encode functional protein. In some embodiments, acell that comprises a disrupted gene does not express (e.g., at the cellsurface) a detectable level (e.g. by antibody, e.g., by flow cytometry)of the protein encoded by the gene. A cell that does not express adetectable level of the protein may be referred to as a knockout cell.For example, a cell having a β2M gene edit may be considered a β2Mknockout cell if β2M protein cannot be detected at the cell surfaceusing an antibody that specifically binds β2M protein.

In some embodiments, a disrupted gene may be described as comprising amutated fragment relative to the wild-type counterpart. The mutatedfragment may comprise a deletion, a nucleotide substitution, anaddition, or a combination thereof. In other embodiments, a disruptedgene may be described as having a deletion of a fragment that is presentin the wild-type counterpart. In some instances, the 5′ end of thedeleted fragment may be located within the gene region targeted by adesigned guide RNA such as those disclosed herein (known as on-targetsequence) and the 3′ end of the deleted fragment may go beyond thetargeted region. Alternatively, the 3′ end of the deleted fragment maybe located within the targeted region and the 5′ end of the deletedfragment may go beyond the targeted region.

In some instances, the disrupted TRAC gene in the anti-CD19 CAR-T cellsdisclosed herein may comprise a deletion, for example, a deletion of afragment in Exon 1 of the TRAC gene locus. In some examples, thedisrupted TRAC gene comprises a deletion of a fragment comprising thenucleotide sequence of SEQ ID NO: 26, which is the target site of TRACguide RNA TA-1. See sequence table below. In some examples, the fragmentof SEQ ID NO: 26 may be replaced by a nucleic acid encoding theanti-CD19 CAR. Such a disrupted TRAC gene may comprise the nucleotidesequence of SEQ ID NO: 39.

The disrupted B2M gene in the anti-CD19 CAR-T cells disclosed herein maybe generated using the CRISPR/Cas technology. In some examples, a B2MgRNA provided in the sequence table below can be used. The disrupted B2Mgene may comprise a nucleotide sequence of any one of SEQ ID Nos: 9-14.

(iii) Exemplary Population of Anti-CD19 CAR-T Cells for AllogeneicTherapy

Also provided herein is population of genetically engineered immunecells (e.g., T cells such as human T cells) comprising the anti-CD19CAR-T cells disclosed herein, which express any of the anti-CD19 CARdisclosed herein (e.g., the anti-CD19 CAR comprising the amino acidsequence of SEQ ID NO: 40), and a disrupted TRAC gene and/or a disruptedB2M gene as also disclosed herein. In some examples, the population ofgenetically engineered T cells are CTX110 cells, which are CD19-directedT cells having disrupted TRAC gene and B2M gene. The nucleic acidencoding the anti-CD19 CAR can be inserted in the disrupted TRAC gene atthe site of SEQ ID NO: 26, which is replaced by the nucleic acidencoding the anti-CD19 CAR, thereby disrupting expression of the TRACgene. The disrupted TRAC gene in the CTX110 cells may comprise thenucleotide sequence of SEQ ID NO: 39.

CTX110 cells can be produced via ex vivo genetic modification using theCRISPR/Cas9 (Clustered Regularly Interspaced Short PalindromicRepeats/CRISPR associated protein 9) technology to disrupt targetedgenes (TRAC and B2M genes), and adeno-associated virus (AAV)transduction to deliver the anti-CD19 CAR construct.CRISPR-Cas9-mediated gene editing involves two guide RNAs (sgRNAs): TA-1sgRNA (SEQ ID NO: 18), which targets the TRAC locus, and B2M-1 sgRNA(SEQ ID NO: 20), which targets the β2M locus. For any of the gRNAsequences provided herein, those that do not explicitly indicatemodifications are meant to encompass both unmodified sequences andsequences having any suitable modifications.

The anti-CD19 CAR of CTX110 cells is composed of an anti-CD19single-chain antibody fragment (scFv, which may comprise the amino acidsequence of SEQ ID NO: 47), followed by a CD8 hinge and transmembranedomain (e.g., comprising the amino acid sequence of SEQ ID NO: 32) thatis fused to an intracellular co-signaling domain of CD28 (e.g., SEQ IDNO: 36) and a CD3ζ signaling domain (e.g., SEQ ID NO: 38). In specificexamples, the anti-CD19 CAR in CTX110 cells comprises the amino acidsequence of SEQ ID NO:40.

In some embodiments, at least 30% of a population of CTX110 cellsexpress a detectable level of the anti-CD19 CAR. For example, at least40%, at least 50%, at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% of the CTX110 cellsexpress a detectable level of the anti-CD19 CAR.

In some embodiments, at least 50% of a population of CTX110 cells maynot express a detectable level of β2M surface protein. For example, atleast 55%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% of the CTX110 cells may notexpress a detectable level of β2M surface protein. In some embodiments,50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%,60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or90%-100% of the engineered T cells of a population does not express adetectable level of β2M surface protein.

Alternatively or in addition, at least 50% of a population of CTX110cells may not express a detectable level of TCR surface protein. Forexample, at least 55%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% of the CTX110cells may not express a detectable level of TCR surface protein. In someembodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%,60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%,80%-90%, or 90%-100% of the engineered T cells of a population does notexpress a detectable level of TRAC surface protein. In specificexamples, more than 90% (e.g., more than 99.5%) of the CTX110 cells donot express a detectable TCR surface protein.

In some embodiments, a substantial percentage of the population ofCTX110 T cells may comprise more than one gene edit, which results in acertain percentage of cells not expressing more than one gene and/orprotein.

For example, at least 50% of a population of CTX110 cells may notexpress a detectable level of two surface proteins, e.g., does notexpress a detectable level of $2M and TRAC proteins. In someembodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%,60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%,80%-90%, or 90%-100% of the CTX110 T cells do not express a detectablelevel of TRAC and B2M surface proteins. In another example, at least 50%of a population of the CTX110 cells do not express a detectable level ofTRAC and B2M surface proteins.

In some embodiments, the population of CTX110 T cells may comprise morethan one gene edit (e.g., in more than one gene), which may be an editdescribed herein. For example, the population of CTX110 T cells maycomprise a disrupted TRAC gene via the CRISPR/Cas technology using theTA-1 TRAC gRNA. In some examples, the CTX110 cells may comprise adeletion in the TRAC gene relative to unmodified T cells. For example,the CTX110 T cells may comprise a deletion of the fragmentAGAGCAACAGTGCTGTGGCC (SEQ ID NO: 26) in the TRAC gene. This fragment canbe replaced by the nucleic acid encoding the anti-CD19 CAR (e.g., SEQ IDNO: 39). Alternatively or in addition, the population of CTX110 cellsmay comprise a disrupted β2M gene via CRISPR/Cas9 technology using thegRNA of B2M-1. Such CTX110 cells may comprise Indels in the β2M gene,which comprise one or more of the nucleotide sequences of SEQ ID NOs:9-14. In specific examples, CTX110 cells comprise ≥30% CAR T cells, ≤50%B2M⁺ cells, and ≤30% TCRαβ⁺ cells. In additional specific examples,CTX110 cells comprise ≥30% CAR⁺ T cells, ≤30% B2M⁺ cells, and ≤0.5%TCRαβ⁺ cells.

See also WO 2019/097305A2, and WO2019215500, the relevant disclosures ofeach of which are incorporated by reference for the subject matter andpurpose referenced herein.

(iv) Pharmaceutical Compositions

In some aspects, the present disclosure provides pharmaceuticalcompositions comprising any of the populations of genetically engineeredanti-CD19 CAR T cells as disclosed herein, for example, CTX110 cells,and a pharmaceutically acceptable carrier. Such pharmaceuticalcompositions can be used in cancer treatment in human patients, which isalso disclosed herein.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of the subject withoutexcessive toxicity, irritation, allergic response, or other problems orcomplications commensurate with a reasonable benefit/risk ratio. As usedherein, the term “pharmaceutically acceptable carrier” refers tosolvents, dispersion media, coatings, antibacterial agents, antifungalagents, isotonic and absorption delaying agents, or the like that arephysiologically compatible. The compositions can include apharmaceutically acceptable salt, e.g., an acid addition salt or a baseaddition salt. See, e.g., Berge et al., (1977) J Pharm Sci 66:1-19.

In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable salt. Non-limiting examples ofpharmaceutically acceptable salts include acid addition salts (formedfrom a free amino group of a polypeptide with an inorganic acid (e.g.,hydrochloric or phosphoric acids), or an organic acid such as acetic,tartaric, mandelic, or the like). In some embodiments, the salt formedwith the free carboxyl groups is derived from an inorganic base (e.g.,sodium, potassium, ammonium, calcium or ferric hydroxides), or anorganic base such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, or the like).

In some embodiments, the pharmaceutical composition disclosed hereincomprises a population of the genetically engineered anti-CD19 CAR-Tcells (e.g., CTX110 cells) suspended in a cryopreservation solution(e.g., CryoStor® C55). The cryopreservation solution for use in thepresent disclosure may also comprise adenosine, dextrose, dextran-40,lactobionic acid, sucrose, mannitol, a buffer agent such asN-)2-hydroxethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), one ormore salts (e.g., calcium chloride, magnesium chloride, potassiumchloride, potassium bicarbonate, potassium phosphate, etc.), one or morebase (e.g., sodium hydroxide, potassium hydroxide, etc.), or acombination thereof. Components of a cryopreservation solution may bedissolved in sterile water (injection quality). Any of thecryopreservation solution may be substantially free of serum(undetectable by routine methods).

In some instances, a pharmaceutical composition comprising a populationof genetically engineered anti-CD19 CAR-T cells such as the CTX110 cellssuspended in a cryopreservation solution (e.g., substantially free ofserum) may be placed in storage vials.

Any of the pharmaceutical compositions disclosed herein, comprising apopulation of genetically engineered anti-CD19 CAR T cells as alsodisclosed herein (e.g., CTX110 cells), which optionally may be suspendedin a cryopreservation solution as disclosed herein may be stored in anenvironment that does not substantially affect viability and bioactivityof the T cells for future use, e.g., under conditions commonly appliedfor storage of cells and tissues. In some examples, the pharmaceuticalcomposition may be stored in the vapor phase of liquid nitrogen at≤−135° C. No significant changes were observed with respect toappearance, cell count, viability, % CAR⁺ T cells, % TCR⁺ T cells, and %B2M⁺ T cells after the cells have been stored under such conditions fora period of time.

II. Preparation of Genetically Engineered Immune Cells

Any suitable gene editing methods known in the art can be used formaking the genetically engineered immune cells (e.g., T cells such asCTX110 cells) disclosed herein, for example, nuclease-dependent targetedediting using zinc-finger nucleases (ZFNs), transcription activator-likeeffector nucleases (TALENs), or RNA-guided CRISPR-Cas9 nucleases(CRISPR/Cas9; Clustered Regular Interspaced Short Palindromic RepeatsAssociated 9). In specific examples, the genetically engineered immunecells such as CTX110 cells are produced by the CRISPR technology incombination with homologous recombination using an adeno-associatedviral vector (AAV) as a donor template.

(1) CRISPR-Cas9-Mediated Gene Editing System

The CRISPR-Cas9 system is a naturally-occurring defense mechanism inprokaryotes that has been repurposed as an RNA-guided DNA-targetingplatform used for gene editing. It relies on the DNA nuclease Cas9, andtwo noncoding RNAs, crisprRNA (crRNA) and trans-activating RNA(tracrRNA), to target the cleavage of DNA. CRISPR is an abbreviation forClustered Regularly Interspaced Short Palindromic Repeats, a family ofDNA sequences found in the genomes of bacteria and archaea that containfragments of DNA (spacer DNA) with similarity to foreign DNA previouslyexposed to the cell, for example, by viruses that have infected orattacked the prokaryote. These fragments of DNA are used by theprokaryote to detect and destroy similar foreign DNA uponre-introduction, for example, from similar viruses during subsequentattacks. Transcription of the CRISPR locus results in the formation ofan RNA molecule comprising the spacer sequence, which associates withand targets Cas (CRISPR-associated) proteins able to recognize and cutthe foreign, exogenous DNA. Numerous types and classes of CRISPR/Cassystems have been described (see, e.g., Koonin et al., (2017) Curr OpinMicrobiol 37:67-78).

crRNA drives sequence recognition and specificity of the CRISPR-Cas9complex through Watson-Crick base pairing typically with a 20 nucleotide(nt) sequence in the target DNA. Changing the sequence of the 5′ 20 ntin the crRNA allows targeting of the CRISPR-Cas9 complex to specificloci. The CRISPR-Cas9 complex only binds DNA sequences that contain asequence match to the first 20 nt of the crRNA, if the target sequenceis followed by a specific short DNA motif (with the sequence NGG)referred to as a protospacer adjacent motif (PAM).

TracrRNA hybridizes with the 3′ end of crRNA to form an RNA-duplexstructure that is bound by the Cas9 endonuclease to form thecatalytically active CRISPR-Cas9 complex, which can then cleave thetarget DNA.

Once the CRISPR-Cas9 complex is bound to DNA at a target site, twoindependent nuclease domains within the Cas9 enzyme each cleave one ofthe DNA strands upstream of the PAM site, leaving a double-strand break(DSB) where both strands of the DNA terminate in a base pair (a bluntend).

After binding of CRISPR-Cas9 complex to DNA at a specific target siteand formation of the site-specific DSB, the next key step is repair ofthe DSB. Cells use two main DNA repair pathways to repair the DSB:non-homologous end joining (NHEJ) and homology-directed repair (HDR).

NHEJ is a robust repair mechanism that appears highly active in themajority of cell types, including non-dividing cells. NHEJ iserror-prone and can often result in the removal or addition of betweenone and several hundred nucleotides at the site of the DSB, though suchmodifications are typically <20 nt. The resulting insertions anddeletions (indels) can disrupt coding or noncoding regions of genes.Alternatively, HDR uses a long stretch of homologous donor DNA, providedendogenously or exogenously, to repair the DSB with high fidelity. HDRis active only in dividing cells, and occurs at a relatively lowfrequency in most cell types. In many embodiments of the presentdisclosure, NHEJ is utilized as the repair operant.

(a) Cas9

In some embodiments, the Cas9 (CRISPR associated protein 9) endonucleaseis used in a CRISPR method for making the genetically engineered T cellsas disclosed herein. The Cas9 enzyme may be one from Streptococcuspyogenes, although other Cas9 homologs may also be used. It should beunderstood, that wild-type Cas9 may be used or modified versions of Cas9may be used (e.g., evolved versions of Cas9, or Cas9 orthologues orvariants), as provided herein. In some embodiments, Cas9 comprises aStreptococcus pyogenes-derived Cas9 nuclease protein that has beenengineered to include C- and N-terminal SV40 large T antigen nuclearlocalization sequences (NLS). The resulting Cas9 nuclease(sNLS-spCas9-sNLS) is a 162 kDa protein that is produced by recombinantE. coli fermentation and purified by chromatography. The spCas9 aminoacid sequence can be found as UniProt Accession No. Q99ZW2, which isprovided herein as SEQ ID NO: 55.

(b) Guide RNAs (gRNAs)

CRISPR-Cas9-mediated gene editing as described herein includes the useof a guide RNA or a gRNA. As used herein, a “gRNA” refers to agenome-targeting nucleic acid that can direct the Cas9 to a specifictarget sequence within a TRAC gene or a β2M gene for gene editing at thespecific target sequence. A guide RNA comprises at least a spacersequence that hybridizes to a target nucleic acid sequence within atarget gene for editing, and a CRISPR repeat sequence.

An exemplary gRNA targeting a TRAC gene is provided in SEQ ID NO: 18 or22. See the sequence table below. See also WO 2019/097305A2, therelevant disclosures of which are incorporated by reference herein forthe subject matter and purpose referenced herein. Other gRNA sequencesmay be designed using the TRAC gene sequence located on chromosome 14(GRCh38: chromosome 14: 22,547,506-22,552,154; Ensembl;ENSG00000277734). In some embodiments, gRNAs targeting the TRAC genomicregion and Cas9 create breaks in the TRAC genomic region resultingIndels in the TRAC gene disrupting expression of the mRNA or protein.

An exemplary gRNA targeting a β2M gene is provided in SEQ ID NO: 20 or24. See the sequence table below. See also WO 2019/097305A2, therelevant disclosures of which are incorporated by reference herein forthe purpose and subject matter referenced herein. Other gRNA sequencesmay be designed using the β2M gene sequence located on Chromosome 15(GRCh38 coordinates: Chromosome 15: 44,711,477-44,718,877; Ensembl:ENSG00000166710). In some embodiments, gRNAs targeting the β2M genomicregion and RNA-guided nuclease create breaks in the β2M genomic regionresulting in Indels in the β2M gene disrupting expression of the mRNA orprotein.

In Type II systems, the gRNA also comprises a second RNA called thetracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence andtracrRNA sequence hybridize to each other to form a duplex. In the TypeV gRNA, the crRNA forms a duplex. In both systems, the duplex binds asite-directed polypeptide, such that the guide RNA and site-directpolypeptide form a complex. In some embodiments, the genome-targetingnucleic acid provides target specificity to the complex by virtue of itsassociation with the site-directed polypeptide. The genome-targetingnucleic acid thus directs the activity of the site-directed polypeptide.

As is understood by the person of ordinary skill in the art, each guideRNA is designed to include a spacer sequence complementary to itsgenomic target sequence. See Jinek et al., Science, 337, 816-821 (2012)and Deltcheva et al., Nature, 471, 602-607 (2011).

In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is adouble-molecule guide RNA. In some embodiments, the genome-targetingnucleic acid (e.g., gRNA) is a single-molecule guide RNA.

A double-molecule guide RNA comprises two strands of RNA molecules. Thefirst strand comprises in the 5′ to 3′ direction, an optional spacerextension sequence, a spacer sequence and a minimum CRISPR repeatsequence. The second strand comprises a minimum tracrRNA sequence(complementary to the minimum CRISPR repeat sequence), a 3′ tracrRNAsequence and an optional tracrRNA extension sequence.

A single-molecule guide RNA (referred to as a “sgRNA”) in a Type IIsystem comprises, in the 5′ to 3′ direction, an optional spacerextension sequence, a spacer sequence, a minimum CRISPR repeat sequence,a single-molecule guide linker, a minimum tracrRNA sequence, a 3′tracrRNA sequence and an optional tracrRNA extension sequence. Theoptional tracrRNA extension may comprise elements that contributeadditional functionality (e.g., stability) to the guide RNA. Thesingle-molecule guide linker links the minimum CRISPR repeat and theminimum tracrRNA sequence to form a hairpin structure. The optionaltracrRNA extension comprises one or more hairpins. A single-moleculeguide RNA in a Type V system comprises, in the 5′ to 3′ direction, aminimum CRISPR repeat sequence and a spacer sequence.

The “target sequence” is in a target gene that is adjacent to a PAMsequence and is the sequence to be modified by Cas9. The “targetsequence” is on the so-called PAM-strand in a “target nucleic acid,”which is a double-stranded molecule containing the PAM-strand and acomplementary non-PAM strand. One of skill in the art recognizes thatthe gRNA spacer sequence hybridizes to the complementary sequencelocated in the non-PAM strand of the target nucleic acid of interest.Thus, the gRNA spacer sequence is the RNA equivalent of the targetsequence.

For example, if the TRAC target sequence is 5′-AGAGCAACAGTGCTGTGGCC-3′(SEQ ID NO: 26), then the gRNA spacer sequence is5′-AGAGCAACAGUGCUGUGGCC-3′ (SEQ ID NO: 19). In another example, if theβ2M target sequence is 5′-GCTACTCTCTCTTTCTGGCC-3′ (SEQ ID NO: 27), thenthe gRNA spacer sequence is 5′-GCUACUCUCUCUUUCUGGCC-3′ (SEQ ID NO: 21).The spacer of a gRNA interacts with a target nucleic acid of interest ina sequence-specific manner via hybridization (i.e., base pairing). Thenucleotide sequence of the spacer thus varies depending on the targetsequence of the target nucleic acid of interest.

In a CRISPR/Cas system herein, the spacer sequence is designed tohybridize to a region of the target nucleic acid that is located 5′ of aPAM recognizable by a Cas9 enzyme used in the system. The spacer mayperfectly match the target sequence or may have mismatches. Each Cas9enzyme has a particular PAM sequence that it recognizes in a target DNA.For example, S. pyogenes recognizes in a target nucleic acid a PAM thatcomprises the sequence 5′-NRG-3′, where R comprises either A or G, whereN is any nucleotide and N is immediately 3′ of the target nucleic acidsequence targeted by the spacer sequence.

In some embodiments, the target nucleic acid sequence has 20 nucleotidesin length. In some embodiments, the target nucleic acid has less than 20nucleotides in length. In some embodiments, the target nucleic acid hasmore than 20 nucleotides in length. In some embodiments, the targetnucleic acid has at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30 or more nucleotides in length. In some embodiments, thetarget nucleic acid has at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30 or more nucleotides in length. In some embodiments, thetarget nucleic acid sequence has 20 bases immediately 5′ of the firstnucleotide of the PAM. For example, in a sequence comprising5′-NNNNNNNNNNNNNNNNNNNNNRG-3′, the target nucleic acid can be thesequence that corresponds to the Ns, wherein N can be any nucleotide,and the underlined NRG sequence is the S. pyogenes PAM. Examples areprovides as SEQ ID NOs: 15-17.

The guide RNA disclosed herein may target any sequence of interest viathe spacer sequence in the crRNA. In some embodiments, the degree ofcomplementarity between the spacer sequence of the guide RNA and thetarget sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacersequence of the guide RNA and the target sequence in the target gene is100% complementary. In other embodiments, the spacer sequence of theguide RNA and the target sequence in the target gene may contain up to10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to4, up to 3, up to 2, or up to 1 mismatch.

Non-limiting examples of gRNAs that may be used as provided herein areprovided in WO 2019/097305A2, and WO2019/215500, the relevantdisclosures of each of which are herein incorporated by reference forthe purposes and subject matter referenced herein. For any of the gRNAsequences provided herein, those that do not explicitly indicatemodifications are meant to encompass both unmodified sequences andsequences having any suitable modifications.

The length of the spacer sequence in any of the gRNAs disclosed hereinmay depend on the CRISPR/Cas9 system and components used for editing anyof the target genes also disclosed herein. For example, different Cas9proteins from different bacterial species have varying optimal spacersequence lengths. Accordingly, the spacer sequence may have 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length.In some embodiments, the spacer sequence may have 18-24 nucleotides inlength. In some embodiments, the targeting sequence may have 19-21nucleotides in length. In some embodiments, the spacer sequence maycomprise nucleotides in length.

In some embodiments, the gRNA can be a sgRNA, which may comprise a 20nucleotide spacer sequence at the 5′ end of the sgRNA sequence. In someembodiments, the sgRNA may comprise a less than 20 nucleotide spacersequence at the 5′ end of the sgRNA sequence. In some embodiments, thesgRNA may comprise a more than 20 nucleotide spacer sequence at the 5′end of the sgRNA sequence. In some embodiments, the sgRNA comprises avariable length spacer sequence with 17-30 nucleotides at the 5′ end ofthe sgRNA sequence.

In some embodiments, the sgRNA comprises no uracil at the 3′ end of thesgRNA sequence. In other embodiments, the sgRNA may comprise one or moreuracil at the 3′ end of the sgRNA sequence. For example, the sgRNA cancomprise 1-8 uracil residues, at the 3′ end of the sgRNA sequence, e.g.,1, 2, 3, 4, 5, 6, 7, or 8 uracil residues at the 3′ end of the sgRNAsequence.

Any of the gRNAs disclosed herein, including any of the sgRNAs, may beunmodified. Alternatively, it may contain one or more modifiednucleotides and/or modified backbones. For example, a modified gRNA suchas a sgRNA can comprise one or more 2′-O-methyl phosphorothioatenucleotides, which may be located at either the 5′ end, the 3′ end, orboth.

In certain embodiments, more than one guide RNAs can be used with aCRISPR/Cas nuclease system. Each guide RNA may contain a differenttargeting sequence, such that the CRISPR/Cas system cleaves more thanone target nucleic acid. In some embodiments, one or more guide RNAs mayhave the same or differing properties such as activity or stabilitywithin the Cas9 RNP complex. Where more than one guide RNA is used, eachguide RNA can be encoded on the same or on different vectors. Thepromoters used to drive expression of the more than one guide RNA is thesame or different.

It should be understood that more than one suitable Cas9 and more thanone suitable gRNA can be used in methods described herein, for example,those known in the art or disclosed herein. In some embodiments, methodscomprise a Cas9 enzyme and/or a gRNA known in the art. Examples can befound in, e.g., WO 2019/097305A2, and WO2019/215500, the relevantdisclosures of each of which are herein incorporated by reference forthe purposes and subject matter referenced herein.

(ii) AAV Vectors for Delivery of CAR Constructs to T Cells

A nucleic acid encoding an anti-CD19 CAR construct as disclosed hereincan be delivered to a cell using an adeno-associated virus (AAV). AAVsare small viruses which integrate site-specifically into the host genomeand can therefore deliver a transgene, such as CAR. Inverted terminalrepeats (ITRs) are present flanking the AAV genome and/or the transgeneof interest and serve as origins of replication. Also present in the AAVgenome are rep and cap proteins which, when transcribed, form capsidswhich encapsulate the AAV genome for delivery into target cells. Surfacereceptors on these capsids which confer AAV serotype, which determineswhich target organs the capsids will primarily bind and thus what cellsthe AAV will most efficiently infect. There are twelve currently knownhuman AAV serotypes. In some embodiments, the AAV for use in deliveringthe CAR-coding nucleic acid is AAV serotype 6 (AAV6).

Adeno-associated viruses are among the most frequently used viruses forgene therapy for several reasons. First, AAVs do not provoke an immuneresponse upon administration to mammals, including humans. Second, AAVsare effectively delivered to target cells, particularly whenconsideration is given to selecting the appropriate AAV serotype.Finally, AAVs have the ability to infect both dividing and non-dividingcells because the genome can persist in the host cell withoutintegration. This trait makes them an ideal candidate for gene therapy.

A nucleic acid encoding an anti-CD19 CAR can be designed to insert intoa genomic site of interest in the host T cells. In some embodiments, thetarget genomic site can be in a safe harbor locus.

In some embodiments, a nucleic acid encoding the anti-CD19 CAR (e.g.,via a donor template, which can be carried by a viral vector such as anadeno-associated viral (AAV) vector) can be designed such that it caninsert into a location within a TRAC gene to disrupt the TRAC gene inthe genetically engineered T cells and express the CAR polypeptide.Disruption of TRAC leads to loss of function of the endogenous TCR. Forexample, a disruption in the TRAC gene can be created with anendonuclease such as those described herein and one or more gRNAstargeting one or more TRAC genomic regions. Any of the gRNAs specific toa TRAC gene and the target regions can be used for this purpose, e.g.,those disclosed herein.

In some examples, a genomic deletion in the TRAC gene and replacement bya CAR coding segment can be created by homology directed repair or HDR(e.g., using a donor template, which may be part of a viral vector suchas an adeno-associated viral (AAV) vector). In some embodiments, adisruption in the TRAC gene can be created with an endonuclease as thosedisclosed herein and one or more gRNAs targeting one or more TRACgenomic regions, and inserting a CAR coding segment into the TRAC gene.

A donor template as disclosed herein can contain a coding sequence for aCAR. In some examples, the CAR-coding sequence may be flanked by tworegions of homology to allow for efficient HDR at a genomic location ofinterest, for example, at a TRAC gene using CRISPR-Cas9 gene editingtechnology. In this case, both strands of the DNA at the target locuscan be cut by a CRISPR Cas9 enzyme guided by gRNAs specific to thetarget locus. HDR then occurs to repair the double-strand break (DSB)and insert the donor DNA coding for the CAR. For this to occurcorrectly, the donor sequence is designed with flanking residues whichare complementary to the sequence surrounding the DSB site in the targetgene (hereinafter “homology arms”), such as the TRAC gene. Thesehomology arms serve as the template for DSB repair and allow HDR to bean essentially error-free mechanism. The rate of homology directedrepair (HDR) is a function of the distance between the mutation and thecut site so choosing overlapping or nearby target sites is important.Templates can include extra sequences flanked by the homologous regionsor can contain a sequence that differs from the genomic sequence, thusallowing sequence editing.

Alternatively, a donor template may have no regions of homology to thetargeted location in the DNA and may be integrated by NHEJ-dependent endjoining following cleavage at the target site.

A donor template can be DNA or RNA, single-stranded and/ordouble-stranded, and can be introduced into a cell in linear or circularform. If introduced in linear form, the ends of the donor sequence canbe protected (e.g., from exonucleolytic degradation) by methods known tothose of skill in the art. For example, one or more dideoxynucleotideresidues are added to the 3′ terminus of a linear molecule and/orself-complementary oligonucleotides are ligated to one or both ends.See, for example, Chang et al., (1987) Proc. Natl. Acad. Sci. USA84:4959-4963; Nehls et al., (1996) Science 272:886-889. Additionalmethods for protecting exogenous polynucleotides from degradationinclude, but are not limited to, addition of terminal amino group(s) andthe use of modified internucleotide linkages such as, for example,phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyriboseresidues.

A donor template can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover, adonor template can be introduced into a cell as naked nucleic acid, asnucleic acid complexed with an agent such as a liposome or poloxamer, orcan be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

A donor template, in some embodiments, can be inserted at a site nearbyan endogenous promoter (e.g., downstream or upstream) so that itsexpression can be driven by the endogenous promoter. In otherembodiments, the donor template may comprise an exogenous promoterand/or enhancer, for example, a constitutive promoter, an induciblepromoter, or tissue-specific promoter to control the expression of theCAR gene. In some embodiments, the exogenous promoter is an EF1apromoter. Other promoters may be used.

Furthermore, exogenous sequences may also include transcriptional ortranslational regulatory sequences, for example, promoters, enhancers,insulators, internal ribosome entry sites, sequences encoding 2Apeptides and/or polyadenylation signals.

To prepare the genetically engineered immune cells (e.g., T cellsdisclosed herein), immune cells such as T cells from a suitable sourcemay be obtained, e.g., blood cells from a human donor, who may be ahealthy donor or a patient need CAR-T cell therapy. The CTX110 cells canbe made using blood cells from one or more healthy human donors.Manufacturing from healthy donor cells minimizes the risk ofunintentionally transducing malignant lymphoma/leukemia cells andpotentially may improve the functionality of the CAR T cells. Thecomponents of the CRISPR system (e.g., Cas9 protein and the gRNAs),optionally the AAV donor template, may be delivered into the host immunecells via conventional approaches. In some examples, the Cas9 and thegRNAs can form a ribonucleoprotein complex (RNP), which can be deliveredto the host immune cells by electroporation. Optionally, the AAV donortemplate may be delivered to the immune cells concurrently with the RNPcomplex. Alternatively, delivery of the RNPs and the AAV donor templatecan be performed sequentially. In some examples, the T cells may beactivated prior to delivery of the gene editing components.

After delivery of the gene editing components and optionally the donortemplate, the cells may be recovered and expanded in vitro. Gene editingefficiency can be evaluated using routine methods for confirm knock-inof the anti-CD19 CAR and knock-out of the target genes (e.g., TRAC, B2M,or both). In some examples, TCRαβ⁺ T cells may be removed. Additionalinformation for preparation of the genetically engineered immune cellsdisclosed herein such as the CTX110 cells can be found in U.S. PatentApplication No. 62/934,991, the relevant disclosures of which areincorporated by reference for the purpose and subject matter referencedherein.

III. Allogeneic CAR-T Cell Therapy of B Cell Malignancies

In some aspects, provided herein are methods for treating a humanpatient having a B cell malignancy using a population of any of thegenetically engineered anti-CD19 CAR T cells such as the CTX110 T cellsas disclosed herein. The allogeneic anti-CD19 CART cell therapy maycomprise two stages of treatment: (i) a conditioning regimen(lymphodepleting treatment), which comprises giving one or more doses ofone or more lymphodepleting agents to a suitable human patient, and (ii)a treatment regimen (allogeneic anti-CD19 CAR T cell therapy), whichcomprises administration of the population of allogeneic anti-CD19 CAR Tcells such as the CTX110 T cells as disclosed herein to the humanpatient.

(i) Patient Population

A human patient may be any human subject for whom diagnosis, treatment,or therapy is desired. A human patient may be of any age. In someembodiments, the human patient is an adult (e.g., a person who is atleast 18 years old). In some examples, the human patient may have a bodyweight of 50 kg or higher. In some embodiments, the human patient can bea child.

A human patient to be treated by the methods described herein can be ahuman patient having, suspected of having, or a risk for having a B cellmalignancy. A subject suspected of having a B cell malignancy might showone or more symptoms of B cell malignancy, e.g., unexplained weightloss, fatigue, night sweats, shortness of breath, or swollen glands. Asubject at risk for a B cell malignancy can be a subject having one ormore of the risk factors for B cell malignancy, e.g., a weakened immunesystem, age, male, or infection (e.g., Epstein-Barr virus infection). Ahuman patient who needs the anti-CD19 CAR T cell (e.g., CTX110 T cell)treatment may be identified by routine medical examination, e.g.,physical examination, laboratory tests, biopsy (e.g., bone marrow biopsyand/or lymph node biopsy), magnetic resonance imaging (MRI) scans, orultrasound exams.

Examples of B cell malignancies that may be treated using the methodsdescribed herein include, but are not limited to, diffuse large B celllymphoma (DLBCL), high grade B cell lymphoma with MYC and BCL2 and/orBCL6 rearrangement, transformed follicular lymphoma (FL), grade 3b FL,or Richter's transformation of chronic lymphocytic leukemia (CLL). Insome examples, the B cell malignancy is DLBCL, e.g., high grade DLBCL orDLBCL not otherwise specified (NOS). In some examples, the B cellmalignancy is transformed FL or grade 3b FL. In some examples, the humanpatient has at least one measurable lesion that is fluorodeoxyglucosepositron emission tomography (PET)-positive.

In some embodiment, the human patient to be treated has DLBCL andexhibits pararectal mass, retroperitoneal mass, diffuse lymph nodes(LN), lytic lesions, tonsillar lesion, or a combination thereof.Alternatively or in addition, the human patient may have bone marrowdiffusion. In other examples, the human patient is free of bone marrowdiffusion.

In some embodiments, the human patient to be treated has transformed FL.Such a human patient may exhibit diffuse LN. In some instances, thehuman patient may have bone marrow diffusion. In other instances, thehuman patient may be free of bone marrow diffusion.

A human patient to be treated by methods described herein may be a humanpatient that has relapsed following a treatment and/or that has beenbecome resistant to a treatment and/or that has been non-responsive to atreatment. As used herein, “relapsed” or “relapses” refers to a B cellmalignancy such as those disclosed herein that returns following aperiod of complete response. Progressive disease refers to an instancewhen a disease worsens after the last evaluation (e.g., stable diseaseor partial response). In some embodiments, progression occurs during thetreatment. In some embodiments, relapse occurs after the treatment. Alack of response may be determined by routine medical practice. Forexample, the human patient to be treated by methods described herein maybe a human patient that has had one or more lines of prior anti-cancertherapies. In some instances, the human patient may have undergone twoor more lines of prior anti-cancer therapies, e.g., a chemotherapy, animmunotherapy, a surgery, or a combination thereof. In some examples,the prior anti-cancer therapies may comprise an anti-CD20 antibodytherapy, an anthracycline-containing therapy, or a combination thereof.

In some instances, the human patient has a refractory B cell malignancy.As used herein, “refractory” refers to a B cell malignancy such as thosedisclosed herein that does not respond to or becomes resistant to atreatment. A human patient having a refractory B cell malignancy mayhave progressive disease on last therapy, or has stable diseasefollowing at least two cycles of therapy with duration of stable diseaseof up to 6 months (e.g., up to 5 months, up to 4 months, or up to 3months or up to 2 months or up to 1 month). In some instances, the humanpatient may have undergone a prior autologous hematopoietic stem celltransplantation (HSCT) and showed no response to such (failed) or haveprogressed or relapsed after achieving some response. In otherinstances, the human patient may not be eligible for prior autologousHSCT.

A human patient may be screened to determine whether the patient iseligible to undergo a conditioning regimen (lymphodepleting treatment)and/or an allogeneic anti-CD19 CAR-T cell therapy as disclosed herein.For example, a human patient who is eligible for lymphodepletiontreatment does not show one or more of the following features: (a)significant worsening of clinical status, (b) requirement forsupplemental oxygen to maintain a saturation level of greater than 90%,(c) uncontrolled cardiac arrhythmia, (d) hypotension requiringvasopressor support, (e) active infection, and (f) grade ≥2 acuteneurological toxicity. In another example, a human patient who iseligible for a treatment regimen does not show one or more of thefollowing features: (a) active uncontrolled infection, (b) worsening ofclinical status compared to the clinical status prior to lymphodepletiontreatment, and (c) grade ≥2 acute neurological toxicity.

A human patient may be screened and excluded from the conditioningregimen and/or treatment regimen based on such screening results. Forexample, a human patient may be excluded from a conditioning regimenand/or the allogeneic anti-CD19 CAR-T cell therapy, if the patient meetsone or more of the following exclusion criteria: (a) has an EasternCooperative Oncology Group (ECOG) performance status 0 or 1; (b)adequate renal, liver, cardiac, and/or pulmonary function; (c) free ofprior gene therapy or modified cell therapy; (d) free of prior treatmentcomprising an anti-CD19 antibody; (e) free of prior allogeneic HSCT; (f)free of detectable malignant cells from cerebrospinal fluid; (g) free ofbrain metastases; (h) free of prior central nervous system disorders;(i) free of unstable angina, arrhythmia, and/or myocardial infarction;(j) free of uncontrolled infection; (k) free of immunodeficiencydisorders or autoimmune disorders that require immunosuppressivetherapy; and (1) free of infection by human immunodeficiency virus,hepatitis B virus, or hepatitis C virus.

(ii) Conditioning Regimen (Lymphodepleting Therapy)

Any human patients suitable for the treatment methods disclosed hereinmay receive a lymphodepleting therapy to reduce or deplete theendogenous lymphocyte of the subject.

Lymphodepletion refers to the destruction of endogenous lymphocytesand/or T cells, which is commonly used prior to immunotransplantationand immunotherapy. Lymphodepletion can be achieved by irradiation and/orchemotherapy. A “lymphodepleting agent” can be any molecule capable ofreducing, depleting, or eliminating endogenous lymphocytes and/or Tcells when administered to a subject. In some embodiments, thelymphodepleting agents are administered in an amount effective inreducing the number of lymphocytes by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 96%, 97%, 98%, or at least 99% as comparedto the number of lymphocytes prior to administration of the agents. Insome embodiments, the lymphodepleting agents are administered in anamount effective in reducing the number of lymphocytes such that thenumber of lymphocytes in the subject is below the limits of detection.In some embodiments, the subject is administered at least one (e.g., 2,3, 4, 5 or more) lymphodepleting agents.

In some embodiments, the lymphodepleting agents are cytotoxic agentsthat specifically kill lymphocytes. Examples of lymphodepleting agentsinclude, without limitation, fludarabine, cyclophosphamide, bendamustin,5-fluorouracil, gemcitabine, methotrexate, dacarbazine, melphalan,doxorubicin, vinblastine, cisplatin, oxaliplatin, paclitaxel, docetaxel,irinotecan, etopside phosphate, mitoxantrone, cladribine, denileukindiftitox, or DAB-IL2. In some instances, the lymphodepleting agent maybe accompanied with low-dose irradiation. The lymphodepletion effect ofthe conditioning regimen can be monitored via routine practice.

In some embodiments, the method described herein involves a conditioningregimen that comprises one or more lymphodepleting agents, for example,fludarabine and cyclophosphamide. A human patient to be treated by themethod described herein may receive multiple doses of the one or morelymphodepleting agents for a suitable period (e.g., 1-5 days) in theconditioning stage. The patient may receive one or more of thelymphodepleting agents once per day during the lymphodepleting period.In one example, the human patient receives fludarabine at about 20-50mg/m² (e.g., 30 mg/m²) per day for 2-4 days (e.g., 3 days) andcyclophosphamide at about 500-750 mg/m² (e.g., 500 or 750 mg/m²) per dayfor 2-4 days (e.g., 3 days). In specific examples, the human patient mayreceive fludarabine at about 30 mg/m² and cyclophosphamide at about 500mg/m² per day for three days. In other specific examples, the humanpatient may receive fludarabine at about 30 mg/m² and cyclophosphamideat about 750 mg/m² per day for three days.

The human patient may then be administered any of the anti-CD19 CAR Tcells such as CTX110 cells within a suitable period after thelymphodepleting therapy as disclosed herein. For example, a humanpatient may be subject to one or more lymphodepleting agent about 2-7days (e.g., for example, 2, 3, 4, 5, 6, 7 days) before administration ofthe anti-CD19 CAR+ T cells (e.g., CTX110 cells). In some instances, ahuman patient is administered the anti-CD19 CAR+ T cells (e.g., CTX110cells) within about 4-5 days after the lymphodepleting therapy.

Since the allogeneic anti-CD19 CAR-T cells such as CTX110 cells can beprepared in advance and may be stored at the treatment site, thelymphodepleting therapy as disclosed herein may be applied to a humanpatient having a B cell malignancy within a short time window (e.g.,within 2 weeks) after the human patient is identified as suitable forthe allogeneic anti-CD19 CAR-T cell therapy disclosed herein. Forexample, the first dose of the lymphodepleting therapy (e.g.,fludarabine at about 30 mg/m² and cyclophosphamide at about 500 mg/m² or750 mg/m²) may be administered to the human patient within two weeks(e.g., within 10 days, within 9 days, within 8 days, within 7 days,within 6 days, within 5 days, within 4 days, within 3 days, within twodays, or less) after the human patient is identified as suitable for theallogeneic anti-CD19 CAR-T cell therapy. In some examples, thelymphodepleting therapy may be performed to the human patient within24-72 hours (e.g., within 24 hours) after the human patient isidentified as suitable for the treatment. The patient can then beadministered the CAR-T cells within 2-7 days (e.g., for example, 2, 3,4, 5, 6, or 7 days) after the lymphodepleting treatment. This allows fortimely treatment of the human patient with the allogeneic anti-CD19CAR-T cells disclosed herein such as CTX110 cells after diseasediagnosis and/or patient identification without delay (e.g., delay dueto preparation of the therapeutic cells). In certain instances, apatient may receive the treatment during inpatient hospital care. Incertain instances, a patient may receive the treatment in outpatientcare.

Prior to any of the lymphodepletion steps, a human patient may bescreened for one or more features to determine whether the patient iseligible for lymphodepletion treatment. For example, prior tolymphodepletion, a human patient eligible for lymphodepletion treatmentdoes not show one or more of the following features: (a) significantworsening of clinical status, (b) requirement for supplemental oxygen tomaintain a saturation level of greater than 90%, (c) uncontrolledcardiac arrhythmia, (d) hypotension requiring vasopressor support, (e)active infection, and (f) grade ≥2 acute neurological toxicity.

Following lymphodepletion, a human patient may be screened for one ormore features to determine whether the patient is eligible for treatmentwith anti-CD19 CAR T cells such as the CTX110 cells. For example, priorto anti-CD19 CART cell treatment and after lymphodepletion treatment, ahuman patient eligible for anti-CD19 CAR T cells treatment does not showone or more of the following features: (a) active uncontrolledinfection, (b) worsening of clinical status compared to the clinicalstatus prior to lymphodepletion treatment, and (c) grade ≥2 acuteneurological toxicity.

(iii) Administration of Anti-CD19 CAR T Cells

Administering anti-CD19 CAR T cells may include placement (e.g.,transplantation) of a genetically engineered T cell population asdisclosed herein (e.g., the CTX110 cells) into a human patient as alsodisclosed herein by a method or route that results in at least partiallocalization of the genetically engineered T cell population at adesired site, such as a tumor site, such that a desired effect(s) can beproduced. The genetically engineered T cell population can beadministered by any appropriate route that results in delivery to adesired location in the subject where at least a portion of theimplanted cells or components of the cells remain viable. The period ofviability of the cells after administration to a subject can be as shortas a few hours, e.g., twenty-four hours, to a few days, to several weeksor months, to as long as several years, or even the life time of thesubject, i.e., long-term engraftment. In certain instances, a patientmay receive the genetically engineered T cell population (e.g., CTX110cells) during inpatient hospital care. In certain instances, a patientmay receive genetically engineered T cell population (e.g., CTX110cells) in outpatient care.

For example, in some aspects described herein, an effective amount ofthe genetically engineered T cell population can be administered via asystemic route of administration, such as an intraperitoneal orintravenous route.

In some embodiments, the genetically engineered T cell population isadministered systemically, which refers to the administration of apopulation of cells other than directly into a target site, tissue, ororgan, such that it enters, instead, the subject's circulatory systemand, thus, is subject to metabolism and other like processes. Suitablemodes of administration include injection, infusion, instillation, oringestion. Injection includes, without limitation, intravenous,intramuscular, intra-arterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subarachnoid, intraspinal, intracerebro spinal, and intrasternalinjection and infusion. In some embodiments, the route is intravenous.

An effective amount refers to the amount of a genetically engineered Tcell population needed to prevent or alleviate at least one or moresigns or symptoms of a medical condition (e.g., a B cell malignancy),and relates to a sufficient amount of a genetically engineered T cellpopulation to provide the desired effect, e.g., to treat a subjecthaving a medical condition. An effective amount also includes an amountsufficient to prevent or delay the development of a symptom of thedisease, alter the course of a symptom of the disease (for example butnot limited to, slow the progression of a symptom of the disease), orreverse a symptom of the disease. It is understood that for any givencase, an appropriate effective amount can be determined by one ofordinary skill in the art using routine experimentation.

An effective amount of a genetically engineered T cell population maycomprise about 1×10⁷ CAR+ cells to about 1×10⁹ CAR+ cells, e.g., about1×10⁷ cells to about 1×10⁹ cells that express a CAR that binds CD19(CAR+ cells). In some embodiments, an effective amount of a geneticallyengineered T cell population may comprise at least 1×10⁷ CAR⁺ CTX110cells, at least 3×10⁷ CAR⁺ CTX110 cells, at least 1×10⁸ CAR⁺ CTX110cells, at least 3×10⁸ CAR⁺ CTX110 cells, or at least 1×10⁹ CAR⁺ CTX110cells. In some embodiments, an effective amount of a geneticallyengineered T cell population may comprise a dose of the geneticallyengineered T cell population, e.g., a dose comprising about 1×10⁷ CTX110cells to about 1×10⁹ CTX110 cells.

The efficacy of anti-CD19 CAR T cell therapy described herein can bedetermined by the skilled clinician. An anti-CD19 CART cell therapy(e.g., involving CTX110 cells) is considered “effective”, if any one orall of the signs or symptoms of, as but one example, levels of CD19 arealtered in a beneficial manner (e.g., decreased by at least 10%), orother clinically accepted symptoms or markers of a B cell malignancy areimproved or ameliorated. Efficacy can also be measured by failure of asubject to worsen as assessed by hospitalization or need for medicalinterventions (e.g., progression of the B cell malignancy is halted orat least slowed). Methods of measuring these indicators are known tothose of skill in the art and/or described herein. Treatment includesany treatment of a B cell malignancy in a human patient and includes:(1) inhibiting the disease, e.g., arresting, or slowing the progressionof symptoms; or (2) relieving the disease, e.g., causing regression ofsymptoms; and (3) preventing or reducing the likelihood of thedevelopment of symptoms.

Following each dosing of anti-CD110 CAR T cells, a human patient may bemonitored for acute toxicities such as tumor lysis syndrome (TLS),cytokine release syndrome (CRS), immune effector cell-associatedneurotoxicity syndrome (ICANS), B cell aplasia, hemophagocyticlymphohistiocytosis (HLH), cytopenia, graft-versus-host disease (GvHD),hypertension, renal insufficiency, or a combination thereof.

When a human patient exhibits one or more symptoms of acute toxicity,the human patient may be subjected to toxicity management. Treatmentsfor patients exhibiting one or more symptoms of acute toxicity are knownin the art. For example, a human patient exhibiting a symptom of CRS(e.g., cardiac, respiratory, and/or neurological abnormalities) may beadministered an anti-cytokine therapy. In addition, a human patient thatdoes not exhibit a symptom of CRS may be administered an anti-cytokinetherapy to promote proliferation of anti-CTX110 CAR T cells.

Alternatively, or in addition to, when a human patient exhibits one ormore symptoms of acute toxicity, treatment of the human patient may beterminated. Patient treatment may also be terminated if the patientexhibits one or more signs of an adverse event (AE), e.g., the patienthas an abnormal laboratory finding and/or the patient shows signs ofdisease progression.

The allogeneic anti-CD19 CAR T cell therapy (e.g., involving the CTX110cells) described herein may also be used in combination therapies. Forexample, anti-CD19 CAR T cells treatment methods described herein may beco-used with other therapeutic agents, for treating a B cell malignancy,or for enhancing efficacy of the genetically engineered T cellpopulation and/or reducing side effects of the genetically engineered Tcell population.

IV. Kit for Allogeneic CAR-T Cell Therapy of B Cell Malignancies

The present disclosure also provides kits for use of a population ofanti-CD19 CAR T cells such as CTX110 cells as described herein inmethods for treating a B cell malignancy. Such kits may include one ormore containers comprising a first pharmaceutical composition thatcomprises one or more lymphodepleting agents, and a secondpharmaceutical composition that comprises any nucleic acid or populationof genetically engineered T cells (e.g., those described herein), and apharmaceutically acceptable carrier. Kits comprising the geneticallyengineered CAR-T cells as disclosed herein, such at the CTX110 cells,may be stored and inventoried at the site of care, allowing for rapidtreatment of human patients following diagnosis.

In some embodiments, the kit can comprise instructions for use in any ofthe methods described herein. The included instructions can comprise adescription of administration of the first and/or second pharmaceuticalcompositions to a subject to achieve the intended activity in a humanpatient. The kit may further comprise a description of selecting a humanpatient suitable for treatment based on identifying whether the humanpatient is in need of the treatment. In some embodiments, theinstructions comprise a description of administering the first andsecond pharmaceutical compositions to a human patient who is in need ofthe treatment.

The instructions relating to the use of a population of anti-CD19 CAR Tcells such as CTX110 T cells described herein generally includeinformation as to dosage, dosing schedule, and route of administrationfor the intended treatment. The containers may be unit doses, bulkpackages (e.g., multi-dose packages) or sub-unit doses. Instructionssupplied in the kits of the disclosure are typically writteninstructions on a label or package insert. The label or package insertindicates that the population of genetically engineered T cells is usedfor treating, delaying the onset, and/or alleviating a T cell or B cellmalignancy in a subject.

The kits provided herein are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging, and the like. Also contemplated are packages for use incombination with a specific device, such as an inhaler, nasaladministration device, or an infusion device. A kit may have a sterileaccess port (for example, the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The container may also have a sterile access port. At least oneactive agent in the pharmaceutical composition is a population of theanti-CD19 CAR-T cells such as the CTX110 T cells as disclosed herein.

Kits optionally may provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiment, the disclosure provides articles of manufacture comprisingcontents of the kits described above.

General Techniques

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I.Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.);Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell,eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P.Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis,et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan etal., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons,1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies(P. Finch, 1997); Antibodies: a practice approach (D. Catty, ed., IRLPress, 1988-1989); Monoclonal antibodies: a practical approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); Usingantibodies: a laboratory manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practicalApproach, Volumes I and II (D. N. Glover ed. 1985); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. (1985; Transcription andTranslation (B. D. Hames & S. J. Higgins, eds. (1984; Animal CellCulture (R. I. Freshney, ed. (1986; Immobilized Cells and Enzymes (IRLPress, (1986; and B. Perbal, A practical Guide To Molecular Cloning(1984); F. M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

Example 1: Preparation of CD19 Targeting Allogeneic CAR-T Cells

Allogeneic T cells expressing a chimeric antigen receptor (CAR) specificfor CD19 were prepared from healthy donor peripheral blood mononuclearcells as described in US Publication No. US 2018-0325955, incorporatedherein by reference. Briefly, primary human T cells were firstelectroporated with Cas9 or Cas9:sgRNA ribonucleoprotein (RNP) complexestargeting TRAC (AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 26)) and B2M(GCTACTCTCTCTTTCTGGCC (SEQ ID NO: 27)). The DNA double stranded break atthe TRAC locus was repaired by homology directed repair with anAAV6-delivered DNA template (SEQ ID NO: 56) containing right and lefthomology arms to the TRAC locus flanking a chimeric antigen receptor(CAR) cassette. The CAR comprised a single-chain variable fragment(scFv) derived from a murine antibody specific for CD19, a CD8 hingeregion and transmembrane domain and a signaling domain comprising CD3zand CD28 signaling domains. The amino acid sequence of the CAR, andnucleotide sequence encoding the same, is set forth in SEQ ID NOs: 40and 39, respectively. The gRNAs used in this Example comprise thefollowing spacer sequences: TRAC gRNA spacer (AGAGCAACAGUGCUGUGGCC (SEQID NO: 19)); and B2M gRNA spacer (GCUACUCUCUCUUUCUGGCC (SEQ ID NO: 21)).A population of cells comprising TRAC⁻/β2M⁻/anti-CD19 CAR⁺ T cells arereferred to herein as “TC1 cells” or “CTX110 cells”.

With CRISPR/Cas9 editing technology, high frequency knockout of theconstant region of the TCRα gene (TRAC) with ˜98% reduction of TCRsurface expression in human primary T-cells from healthy donors, whichaims to significantly impair graft-versus-host disease (GVHD), wasachieved. High frequency knockout of the β-2-microglobulin (B2M) genecould also be obtained, which aims to increase persistence in patients,potentially leading to increased potency overall. TRAC/B2M doubleknockout frequencies have been obtained in ˜80% of T cells without anysubsequent antibody-based purification or enrichment. Human T cellsexpressing a CD19-specific CAR from within a disrupted TRAC locus,produced by homology-directed repair using an AAV6-delivered donortemplate, along with knockout of the B2M gene have been consistentlyproduced at a high efficiency. This site-specific integration of the CARprotects against the potential outgrowth of CD3+CAR+ cells, furtherreducing the risk of GVHD, while also reducing the risk of insertionalmutagenesis associated with retroviral or lentiviral deliverymechanisms. These engineered allogeneic CAR-T cells show CD19-dependentT-cell cytokine secretion and potent CD19-specific cancer cell lysis.

The production of allogeneic anti-CD19 CAR-T product (FIG. 1) exhibitedefficiency editing (e.g., greater than 50% TRAC−/B2M−/anti-CD19 CAR+Tcells efficiency) (FIG. 2).

Example 2: Dose Escalation Study to Determine the Efficacy of CAR-TCells in the Subcutaneous Raji Human Burkitt's Lymphoma Tumor XenograftModel in NOG Mice

The efficacy of CD19 targeting CAR-T cells against the subcutaneous RajiHuman Burkitt's Lymphoma tumor xenograft model in NOG mice was evaluatedusing methods employed by Translational Drug Development, LLC(Scottsdale, Ariz.). In brief, 12, 5-8 week old female, CIEA NOG(NOD.Cg-Prkd^(scid)Il2rg^(tm1Sug)/JicTac) mice were individually housedin ventilated microisolator cages, maintained under pathogen-freeconditions, 5-7 days prior to the start of the study. On Day 1 micereceived a subcutaneous inoculation of 5×10⁶ Raji cells/mouse. The micewere further divided into 3 treatment groups as shown in Table 1. On Day8 (7 days post inoculation with the Raji cells), treatment group 2 andgroup 3 received a single 200 μl intravenous dose ofTRAC⁻/B2M⁻/anti-CD19 CAR+ cells (TC1) according to Table 1.

TABLE 1 Treatment groups. Group Raji Cells (s.c.) TC1 Treatment (i.v.) N1 5 × 10⁶ cells/mouse None 4 2 5 × 10⁶ cells/mouse 5 × 10⁶ cells/mouse 43 5 × 10⁶ cells/mouse 1 × 10⁷ cells/mouse 4

Tumor volume and body weight was measured and individual mice wereeuthanized when tumor volume was ≥500 mm³.

By Day 18, the data show a statistically significant decrease in thetumor volume in response to TC1 cells as compared to untreated mice(FIG. 3). The effect on tumor volume was dose-dependent (Table 2); micereceiving higher doses of TC1 cells showed significantly reduced tumorvolume when compared to mice receiving either a lower dose of TC1 cellsor no treatment. An increase in survival was also observed in thetreated group (Table 2).

TABLE 2 Tumor response and survival. Tumor Tumor Group volume (Day 18)volume (Day 20) Survival (Days) N 1 379.6 ± 67.10  482 ± 47.37  20-22 42 214.0 ± 20.73 372.2 ± 78.21  25 4 3 107.5 ± 7.33* 157.1 ± 10.62** 27(end of study) 4 p = 0.007 compared to control (Group 1) **p = 0.0005compared to control (Group 1)

Example 3: Assessment of CD19 Targeting CAR-T Cells Efficacy inIntravenous Disseminated Models in NOG Mice

To further assess the efficacy of TRAC⁻/B2M⁻/anti-CD19 CAR+ cells (TC1),disseminated mouse models were utilized.

Intravenous Disseminated Raji Human Burkitt's Lymphoma Tumor XenograftModel

The Intravenous Disseminated Model (Disseminated Model) using the RajiHuman Burkitt's Lymphoma tumor cell line in NOG mice was used to furtherdemonstrate the efficacy of TC1. Efficacy of TC1 was evaluated in theDisseminated Model using methods employed by Translations DrugDevelopment, LLC (Scottsdale, Ariz.) and described herein. In brief, 24,5-8 week old female CIEA NOG (NOD.Cg-Prkde^(scid)I12rg^(t1Sug)/JicTac)mice were individually housed in ventilated microisolator cages,maintained under pathogen-free conditions, 5-7 days prior to the startof the study. At the start of the study, the mice were divided into 5treatment groups as shown in Table 9. On Day 1 mice in Groups 2-5received an intravenous injection of 0.5×10⁶ Raji cells/mouse. The micewere inoculated intravenously to model disseminated disease. On Day 8 (7days post injection with the Raji cells), treatment Groups 3-5 receiveda single 200 μl intravenous dose of TC1 cells (Table 3).

TABLE 3 Treatment groups. Group Raji Cells (i.v.) TC1 Treatment (i.v.) N1 None None 8 2 0.5 × 10⁶ cells/mouse None 4 3 0.5 × 10⁶ cells/mouse 1 ×10⁶ cells/mouse 4 (~0.5 × 10⁶ CAR-T+ cells) 4 0.5 × 10⁶ cells/mouse 2 ×10⁶ cells/mouse 4 (~1.0 × 10⁶ CAR-T+ cells) 5 0.5 × 10⁶ cells/mouse 4 ×10⁶ cells/mouse 4 (~2.0 × 10⁶ CAR-T+ cells)

During the course of the study mice were monitored daily and body weightwas measured two times weekly. A significant endpoint was the time toperi-morbidity and the effect of T-cell engraftment was also assessed.The percentage of animal mortality and time to death were recorded forevery group in the study. Mice were euthanized prior to reaching amoribund state. Mice may be defined as moribund and sacrificed if one ormore of the following criteria were met:

Loss of body weight of 20% or greater sustained for a period of greaterthan 1 week;

Tumors that inhibit normal physiological function such as eating,drinking, mobility and ability to urinate and or defecate;

Prolonged, excessive diarrhea leading to excessive weight loss (>20%);or

Persistent wheezing and respiratory distress.

Animals were also considered moribund if there was prolonged orexcessive pain or distress as defined by clinical observations such as:prostration, hunched posture, paralysis/paresis, distended abdomen,ulcerations, abscesses, seizures and/or hemorrhages.

Similar to the subcutaneous xenograph model (Example 2), theDisseminated Model revealed a statistically significant survivaladvantage in mice treated with TRAC⁻/B2M⁻/anti-CD19 CAR+ cells (TC1) asshown in FIG. 4, p<0.0001. The effect of TC1 treatment on survival inthe disseminated model was also dose dependent (Table 4).

TABLE 4 Animal survival. Raji TC1 Max Median Group Cells (i.v.)Treatment (i.v.) survival (days) survival (days) 1 No No Max Max 2 YesNo 20 20 3 Yes 1 × 10⁶ cells/mouse 21 21 4 Yes 2 × 10⁶ cells/mouse 25 255 Yes 4 × 10⁶ cells/mouse 32 26

A second experiment was run using the Intravenous Disseminated modeldescribed above.

On Day 1 mice in Groups 2-4 received an intravenous injection of 0.5×10⁶Raji cells/mouse. The mice were inoculated intravenously to modeldisseminated disease. On Day 4 (3 days post injection with the Rajicells), treatment Groups 2-4 received a single 200 μl intravenous doseof TC1 cells per Table 5.

TABLE 5 Treatment groups. Group Raji Cells (i.v.) TC1 Treatment (i.v.) N1 0.5 × 10⁶ cells/mouse None 6 2 0.5 × 10⁶ cells/mouse 0.6 × 10⁶ CAR⁺cells/mouse 7 3 0.5 × 10⁶ cells/mouse 1.2 × 10⁶ CAR⁺ cells/mouse 5 4 0.5× 10⁶ cells/mouse 2.4 × 10⁶ CAR⁺ cells/mouse 5

Again, the Disseminated Model revealed a statistically significantsurvival advantage in mice treated with TRAC⁻/B2M⁻/anti-CD19 CAR+ cells(TC1) as shown in FIG. 5, p=0.0016. The effect of TC1 treatment onsurvival in the disseminated model was also dose dependent (Table 6).

TABLE 6 Animal survival. Raji Max Median Cells TC1 survival survivalGroup (i.v.) Treatment (i.v.) (days) (days) Significance 1 Yes No 20 202 Yes 0.6 × 10⁶ CAR⁺ 35 27 p = 0.005 cells/mouse 3 Yes 1.2 × 10⁶ CAR⁺ 3937 p = 0.016 cells/mouse 4 Yes 2.4 × 10⁶ CAR⁺ 49 46 p = 0.016cells/mouse

Evaluation of Splenic Response to TC1 Treatment

The spleen was collected from mice 2-3 weeks following Raji injectionand the tissue was evaluated by flow cytometry for the persistence ofTC1 cells and eradication of Raji cells in the spleen.

The spleen was transferred to 3 mL of 1×DPBS CMF in a C tube anddissociated using the MACS Octo Dissociator. The sample was transferredthrough a 100 micron screen into a 15 mL conical tube, centrifuged (1700rpm, 5 minutes, ART with brake) and resuspended in 1 mL of 1×DPBS CMFfor counting using the Guava PCA. Bone marrow was centrifuged andresuspended in 1 mL of 1×DPBS CMF for counting using the Guava PCA.Cells were resuspended at a concentration of 10×10⁶ cells/mL in 1×DPBSCMF for flow cytometry staining.

Specimens (50 μL) were added to 1 mL 1× Pharm Lyse and incubated for10-12 minutes at room temperature (RT). Samples were centrifuged andthen washed once with 1×DPBS CMF. Samples were resuspended in 50 μL of1×DPBS and incubated with Human and Mouse TruStain for 10-15 minutes atRT. The samples were washed once with 1 mL 1×DPBS CMF and resuspend in50 μL of 1×DPBS CMF for staining. Surface antibodies were added and thecells incubated for 15-20 minutes in the dark at RT and then washed with1 mL 1×DPBS CMF. Then samples were resuspended in 125 μL of 1×DPBS CMFfor acquisition on the flow cytometer. Cells were stained with thefollowing surface antibody panel:

TABLE 7 Antibody panel. FITC PE APC C3 APCCy7 V421 V510 huCD3 huCD45huCD19 7AAD CD8 CD4 mCD45 (UCHT1) (HI30) (HIB19) (SK1) (RPA-T4) (30-F11)

Cell populations were determined by electronic gating (Pl=totalleukocytes) on the basis of forward versus side scatter. Compensation toaddress spill over from one channel to another was performed uponinitial instrument set up using Ultra Comp Beads from Thermo Fisher. Theflow cytometer was set to collect 10,000 CD45+ events in each tube. Flowcytometric data acquisition was performed using the FACSCantoll™ flowcytometer. Data was acquired using BO FACSDiva™ software (version 6.1.3or 8.0.1). Flow cytometry data analysis was in the form of FlowCytograms, which are graphical representations generated to measurerelative percentages for each cell type.

This example demonstrates that following TC1 cell treatment, thetherapeutically beneficial TRAC⁻/B2M⁻/anti-CD19 CAR+ cells persist inthe spleen and selectively eradicate Raji cells from the tissue (FIG.6A). In addition, treatment with TC1 cells do not exhibit Raji inducedincrease in cell mass (FIG. 6A). Further, FIG. 7 shows that theremaining human cells in spleens of mice treated withTRAC⁻/B2M⁻/anti-CD19 CAR+ cells are CD8+. These CD8+ T cells are alsoCD3 negative proving that persistent T cells in this model remainTCR/CD3 negative and are thus edited.

Intravenous Disseminated Nalm-6 Human Acute Lymphoblastic Leukemia TumorXenograft Model

The Intravenous Disseminated Model (Disseminated Model) using the Nalm-6Human Acute Lymphoblastic Leukemia tumor cell line in NOG mice was usedin to further demonstrate the efficacy of TC1. Efficacy of TC1 wasevaluated in the Disseminated Model using methods employed byTranslations Drug Development, LLC (Scottsdale, Ariz.) and describedherein. In brief, 24, 5-8 week old female CIEA NOG(NOD.Cg-Prkd^(scid)Il2rg^(tm1Sug)/JicTac) mice were individually housedin ventilated microisolator cages, maintained under pathogen-freeconditions, 5-7 days prior to the start of the study. At the start ofthe study, the mice were divided into 5 treatment groups as shown inTable 14. On Day 1 mice in Groups 2-4 received an intravenous injectionof 0.5×10⁶ Nalm6 cells/mouse. The mice were inoculated intravenously tomodel disseminated disease. On Day 4 (3 days post injection with theNalm6 cells), treatment Groups 2-4 received a single 200 μl intravenousdose of TC1 cells per Table 8.

TABLE 8 Treatment groups. Group Nalm6 Cells (i.v.) TC1 Treatment (i.v.)N 1 0.5 × 10⁶ cells/mouse None 6 2 0.5 × 10⁶ cells/mouse 1 × 10⁶ CAR⁺cells/mouse 6 3 0.5 × 10⁶ cells/mouse 2 × 10⁶ CAR⁺ cells/mouse 6 4 0.5 ×10⁶ cells/mouse 4 × 10⁶ CAR⁺ cells/mouse 6

During the course of the study mice were monitored daily and body weightwas measured two times weekly as described above.

Similar to the Raji intravenous disseminated model (above), the Nalm6Model also showed a statistically significant survival advantage in micetreated with TRAC⁻/B2M⁻/anti-CD19 CAR+ cells (TC1) as shown in FIG. 8,p=0.0004. The effect of TC1 treatment on survival in the Nalm6disseminated model was also dose dependent (Table 9).

TABLE 9 Animal survival. Nalm6 TC1 Max Median Cells Treatment survivalSurvival Group (i.v.) (i.v.) (days) (days) Significance 1 Yes No 31 25.52 Yes 1 × 10⁶ CAR⁺ 32 31 p = 0.03  cells/mouse 3 Yes 2 × 10⁶ CAR⁺ 38 36p = 0.0004 cells/mouse 4 Yes 4 × 10⁶ CAR⁺ 52 46 p = 0.0004 cells/mouse

Example 4: Further Assessment of CD19 Targeting CAR-T Cells Efficacy inIntravenous Disseminated Models in NOG Mice

The purpose of this study was to evaluate the anti-tumor activity ofanti-CD19 CAR+ T cells at multiple dose levels against theNalm6-Fluc-GFP acute lymphoblastic leukemia tumor cell line in NOG mice.The mice were inoculated intravenously to model disseminated disease.Significant endpoint was time to pen-morbidity. Bioluminescent imagingwas performed to monitor progression of disseminated disease.

In brief, 6 week old female, CIEA NOG(NOD.Cg-Prkdc^(scid)Il2rg^(tm1Sug)/JicTac) mice were housed inventilated microisolator cages, maintained under pathogen-freeconditions, 5-7 days prior to the start of the study. On Day 1 micereceived an intravenous inoculation of 5×10⁴ Nalm6-Fluc-GFP(Nalm6-Fluc-Neo/eGFP—Puro; Imanis Life Sciences (Rochester, Minn.))cells/mouse. Three (3) days post inoculation with Nalm6-Fluc-GFP cells,the mice were divided into treatment groups and dosed with T cellpopulations comprising TRAC−/B2M−/anti-CD19 CAR+ T cells, as indicatedin Table 10. Region of Interest values (ROI) values were captured andreported. Body weight was measured twice daily and bioluminescence wasmeasured twice weekly starting on Day 4 (3 Days Post inoculation ofNalm6-Fluc-GFP cells) through Day 67, once weekly starting Day 74 tostudy end. To measure bioluminescence mice were injectedintraperitoneally with 200 μl of D-Luciferin 150 mg/kg. Kinetics imageswere taken at the beginning of the study and as needed throughout todetermine optimal post D-Luciferin dose and exposure time to image themice. Mice were imaged by capturing luminescence signal (open emission)using an AMI 1000 imaging unit with software version 1.2.0 (SpectralInstruments Imaging Inc.; Tucson, Ariz.).

TABLE 10 Treatment groups. # of T Cells Anti-CD19 Group Anti-CD19 CAR TCell injected (iv) CAR+ T cells N 1 N/A N/A N/A 5 2 TRAC-/β2M-/anti-CD19 3 × 10⁶ ~1.8 × 10⁶ 5 cells/mouse 3 TRAC-/β2M-/anti-CD19  6 × 10⁶ ~3.6 ×10⁶ 5 cells/mouse 4 TRAC-/β2M-/anti-CD19 12 × 10⁶   7.2 × 10⁶ 4cells/mouse

Individual mice were euthanized at pen-morbidity (clinical signssuggesting high tumor burden (e.g., lack of motility, hunch back,hypoactivity) or 20% or greater body weight loss sustained for a periodof greater than 1-week). Mice were euthanized prior to reaching amoribund state. The study was ended on Day 99 when the final mouse waseuthanized as a long-term survivor.

FIG. 9 shows prolonged survival of mice that received different doses ofTC1 cells relative to untreated mice. FIG. 10 shows low to undetectablelevels of bioluminescence in mice that received the highest dose of TC1cells (12×10⁶ cells/mouse) and which resulted in the longest survival asshown in FIG. 9. At day 74 bioluminescence was detected in all 4 mice,indicative of tumor cell expansion in the treatment group.

Overall, these results show a single injection of TC1 cells can prolongsurvival of mice that were administered a lethal dose of Nalm6 B-ALLcells. This prolonged survival is dose dependent with a graded survivalresponse observed between low, middle and high doses of TC1 cells.

Example 5: Analysis of Graft Versus Host Disease in Mice AdministeredAllogeneic CD19 Targeting CAR T Cells

A study in mice was conducted to evaluate the potential for bothunedited human T cells and TC1 cells to cause graft versus host disease(GvHD). After total body irradiation with 200 cGy, NOG female mice wereadministered a single intravenous slow bolus injection of unedited humanT cells or TC1 cells. Animals were followed for up to 119 days afterradiation only (Group 1) or radiation plus a single dose administrationof PBMCs (Group 2), electroporated T cells (Group 3) or TC1 cells (Group4). Cells were administered approximately 6 hours post radiation onDay 1. Table 11 summarizes the groups and study design.

TABLE 11 Treatment groups. Total Number of Group Dose Level IrradiationAnimals Number Test Article (cells/mouse) Dose (Female) 1 Radiation Only0 200 cGy 12 2 Radiation + PBMCs 6 × 10P⁶ 6 3 Radiation + EP T cells 3 ×10P⁷ 6 4 Radiation + TC1 cells 3 × 10P⁷ 6

The endpoints of the study were survival, kinetics of appearance of GvHDsymptoms, and body weight measurements.

Mortality was observed in Group 1 (3 of 12 animals), Group 2 (6 of 6animals) and Group 3 (2 of 6 animals) during the first 30 dayspost-treatment (FIG. 11). All animals in Group 4 (TC1 cells) surviveduntil scheduled necropsy (FIG. 11). Moribund animals in Groups 1, 2 and3 experienced weight loss and/or clinical observations consistent withthe development of GvHD (slight to severe cold to touch, slight tomoderate emaciation, slight to marked hunched posture, severe weightloss, mild to severe alopecia, severe hypoactivity, moderate laboredrespiration, and marked tachypnea). Animals in Groups 1 and 4, andnon-moribund animals in Group 3, experienced mild weight loss followingradiation which improved over the course of the study (FIG. 12). Nonotable clinical observations were recorded.

This study demonstrated that unedited human PBMCs induce fatal GvHD inirradiated NOG mice in all animals (Group 2), with onset 2 to 3 weeksafter administration of cells. In contrast, no mice that received TC1cells (Group 4) developed GvHD during the study (119 days), despite thehigher number of cells that were administered to these animals (3×10⁷TC1 cells per mouse compared to 6×10⁶ PBMCs per mouse). The irradiationprocedure induced transient weight loss in all groups and recovered inall groups that did not receive unedited PBMCs.

A second study was conducted to further evaluate the potential for bothunedited human T cells and TC1 cells to cause GvHD. Specifically,NOD/SCID/IL2Rγnull (NSG) female mice were administered a singleintravenous slow bolus injection of unedited human T cells or TC1 cellsafter a total body irradiation (total irradiation dose of 200 cGy, 160cGy/min; targeted LDR_(0/140)R). The endpoints of this study weresurvival, kinetics of appearance of symptoms of GvHD and body weightmeasurements. Histopathology was also performed on all collectedtissues. Exposure was assessed in mouse blood and tissues by flowcytometry and immunohistochemistry (IHC), where appropriate.

The cells were administered as a single dose via intravenous slow bolusas described in Table 12.

TABLE 12 Study Design. Concen- Total Number Dose tration Ir- of Group(Cells/ (Cells/ radiation Animals Number Test Article Mouse) mL) Dose MF 1 Vehicle - no RT^(a) 0 0  0 cGy  5  5 2 Vehicle - RT^(a) 0 0 200 cGy15 15 3 Unedited T cells 1 × 10⁷  4 × 10⁷ 15 15 4 TC1 - low dose 2 × 10⁷ 8 × 10⁷ 15 15 5 TC1 - high dose 4 × 10⁷ 16 × 10⁷ 15 15 ^(a)Group 1animals were not irradiated and were not dosed with cells (animals wereadministered with vehicle, PBS 1X). Group 2 animals were irradiated butwere not dosed with cells (animals were administered with vehicle,phosphate-buffered saline [PBS]).

Animals were randomized into treatment groups by body weight using avalidated preclinical software system (Provantis). Due to the large sizeof this study, dosing and necropsy activities were staggered over ninedays. To minimize bias, animals from the control and TC1 groups (Groups4 and 5) were dosed and necropsied on the same day. Necropsy occurred onStudy Day 85 for all groups.

Mortality was observed for all animals that received unedited human Tcells (Group 3), with onset at Day 14 (FIG. 13). All mice that receivedunedited human T cells (Group 3), were either found dead or sent tounscheduled euthanasia by Day 29. Clinical signs in these animals wereconsistent with the development of GvHD and included dull fur, slight tosevere decreased activity, hunched back posture, slight to moderatethinness, and increased respiratory rate. Marked changes in hematologyparameters were observed at euthanasia in mice that received uneditedhuman T cells (Group 3), including decreases in red blood cells,hemoglobin, platelets, white blood cells and reticulocyte counts.Minimal to moderate inflammation was observed in the liver, lung,kidney, spleen, and thymus of Group 3 animals. Necrosis oftenaccompanied inflammation in these tissues. These findings wereconsistent with the development of GvHD. Additionally, mild to severehypocellularity in the femoral and sternal bone marrow was also presentin the majority of Group 3 animals, which was likely attributable to theeffects of total body irradiation. This was likely only observed in thisgroup due to the early necropsy dates (2-4 weeks post-radiation),compared to 12 weeks for all other groups. Consistent with the presenceof GvHD, immunohistochemical analysis of Group 3 animals revealed thepresence of human CD45P⁺P cells in all tissues examined (kidney, liver,spleen, lung, skin, and the digestive tract). All animals in the otherGroups survived until the scheduled necropsy.

Further, no significant weight loss was observed in Groups 1, 2, 4, or 5(FIG. 14). No notable clinical observations that were consistent withGvHD, characterized by observations of at least two symptoms consideredlikely to denote GvHD, were recorded in these groups. Several animalsfrom Groups 4 and 5 exhibited symptoms such as dull fur, slight tomoderate decreased activity, and/or slight thinness throughout thestudy. Although these symptoms are often associated with GvHD, they didnot appear to be TC1-related as they were infrequently observed,transient and of short duration, and were also seen in some irradiatedcontrol animals (Group 2).

Overall the results from these two studies confirmed TC1 cells do notinduce graft versus host disease.

Example 6: Preparation and Characterization of Developmental Lots ofAllogeneic CD19 Targeting CAR T Cells

TC1 cells for the purposes of the clinical study were prepared fromhealthy donor peripheral blood mononuclear cells obtained via a standardleukopheresis procedure. The mononuclear cells were enriched for T cellsand activated with anti-CD3/CD28 antibody-coated beads, thenelectroporated with CRISPR-Cas9 ribonucleoprotein complexes andtransduced with a CAR gene-containing recombinant adeno-associated virus(AAV) vector. The modified T cells were expanded in cell culture,purified, formulated into a suspension, and cryopreserved.

Prior to modifying the cells, T cells from six different healthy donorswere evaluated for expression of various cell surface markers.CD27+CD45RO− T cells within the CD8+ subset were previously shown tocorrelate with complete responses in chronic lymphocytic leukemia (CLL)when treated with anti-CD19 CAR T cell therapy (Fraietta et al., NatMed, Vol. 24(5): 563-571, 2018). Accordingly, the percent of CD27+CD45O−T cells within the CD8+ subset of six different donors was evaluated byflow cytometry. In brief, 1×10⁶ cells were incubated with Fab-Biotin orIgG-Biotin antibodies as a negative control. Cells were washed withstaining buffer and incubated with mouse anti-IgG to capture excessprimary antibodies. Cells were washed again and incubated with the fullpanel of secondary antibodies (CD8, Biolegend: Catalog #300924, CD45RO,Biolegend: Catalog #304230, CD27, Biolegend: Catalog #560612) andviability dye. Cells were washed a final time with staining buffer andrun on the flow cytometer to capture various stained populations. FIG.shows the levels of CD27+CD45RO− T cells within their CD8+ subsets.Allogeneic CAR-T manufacturing allows for the selection of donor inputmaterial with favorable characteristics, such as high CD27+CD45RO− cellsin the CD8+ fraction of a donor of interest.

More specifically, leukopaks from 18 to 40 year-old male donors wereused to isolate CD4+ and CD8+ T cells. After isolation, enrichment andactivation of CD4+ and CD8+ T cells, cells were electroporated withribonucleoprotein complexes comprising Cas9 nuclease protein, TRAC sgRNA(SEQ ID NO: 26) or B2M sgRNA (SEQ ID NO: 27). The TRAC and B2Mribonucleoprotein complexes were combined prior to electroporation.After electroporation, freshly thawed rAAV comprising a donor template(SEQ ID NO: 54) encoding the anti-CD19 CAR (SEQ ID NO: 40) was added tothe cells, and cells were incubated. Cells were then expanded in cultureand supplemented with rhIL-2 and rhIL-7 every three to four days. Cellsset up for monitoring were tested for T cell identity and gene editingwith a TCR panel (CD5, CD4, CD8, TCRαβ, B2M and CD45). Upon confirmationof T cell identity, TCRαβ depletion was performed by incubating thecells with a biotin-conjugated anti-TCRαβ antibody and anti-biotinbeads. The depleted cells were recovered and formulated foradministration. The resulting population of cells had less than 0.5%TCRαβ+ cells. FIG. 16 shows the analysis of TCRαβ+ cells before andafter purification.

Eight development lots of TC1 cells were tested for T cell identity.Average results from eight tested lots showed 84.58% knock-out of B2M(i.e., 15.42% B2M⁺ cells) and 99.98% of cells were TCR− (i.e., 0.2%TCR+), and ˜50% knock-in of anti-CD19 CAR (FIG. 17).

In addition, exhaustion and senescent markers were evaluated in donorsbefore and after T cell editing. Specifically, the percentage of PD1+,LAG3+, TIM3+ and CD57+ cells were determined from total T cellpopulations. Expression of the markers was assessed by flow cytometry,as described above, using the following secondary antibodies: MouseAnti-PD1 PeCy7, Biolegend, Catalog #329918; Mouse Anti-TIM3BV421,Biolegend, Catalog #345008; Mouse Anti-CD57 PerCp Cy5.5, Biolegend,Catalog #359622; and Mouse Anti-LAG3 PE, Biolegend, Catalog #369306.FIG. 18 shows that exhaustion or senescent markers never increased over15% of the total T cell population after genome editing.

In addition, selective killing by three different lots of TC1 cells wasevaluated in vitro. Specifically, TC1 cells were incubated withCD19-positive cell lines (K562-CD19; Raji; and Nalm6), or aCD19-negative cell line (K562). Killing was measured using a flowcytometry-based cytotoxicity assay after ˜24 hours. Specifically, targetcells were labeled with 5 μM efluor670 (Thermo Fisher Scientific,Waltham, Mass.), washed and incubated overnight (50,000 targetcells/well; 96-well U-bottom plate [Corning, Tewksbury, Mass.]) inco-cultures with TC1 or control T cells at varying ratios (from 0.1:1 upto 4:1 T cells to target cells). The next day, wells were washed andmedia was replaced with 200 μL of fresh media containing a 1:500dilution of 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Thermo FisherScientific, Waltham, Mass.) to enumerate dead/dying cells. Finally, 25μL of CountBright beads (Thermo Fisher Scientific) was added to eachwell, and cells were then analyzed by flow cytometry using a Novocyteflow cytometer (ACEA Biosciences, San Diego, Calif.). Flowjo software(v10, Flowjo, Ashland, Oreg.) was used to analyze flow cytometry datafiles (fcs files). TCRαβ+ T cells (unedited cells) were used ascontrols. TC1 cells efficiently killed CD19-positive cells at higherrates than unedited T cells, and CD19-negative cells showed low levelsof cell lysis in the presence of TC1 cells that were no more than whenco-cultured with unedited T cells (FIG. 19).

TC1 cells produced from three unique donors were also used to assessgrowth in the absence of cytokine and/or serum. Specifically, TC1 cellswere grown in full T cell media for 14 days. On Day 0, cells fromculture were grown either in complete T-cell media (containing X-VIVO 15(Lonza, Basel, Switzerland), 5% human AB serum (Valley Biomedical,Winchester, Va.), IL-2 (Miltenyi, Bergisch Gladbach, Germany) and IL-7(Cellgenix, Frieburg, Germany)) (Complete Media), media containing serumbut no IL-2 or IL-7 cytokines (5% serum, no cytokines), or no serum orcytokines (No serum, No Cytokines). Cells were enumerated as above forup to 35 days after removal of cytokines and/or serum. No outgrowth ofTC1 cells was observed in the absence of cytokine and/or serum (FIG.20).

For administration, TC1 cells are resuspended in cryopreservativesolution (CryoStor CS-5) and supplied in a 6 mL infusion vial. The totaldose is contained in one or more vials. The infusion of each vial occurswithin 20 minutes of thawing.

Example 7: A Phase I, Open-Label, Multicenter, Dose Escalation andCohort Expansion Study of the Safety and Efficacy of AllogeneicCRISPR-Cas9 Engineered T Cells (CTX110) in Subjects with Relapsed orRefractory B Cell Malignancies

CTX110 is a CD19-directed chimeric antigen receptor (CAR) T cellimmunotherapy comprised of allogeneic T cells that are geneticallymodified ex vivo using CRISPR-Cas9 (clustered regularly interspacedshort palindromic repeats/CRISPR-associated protein 9) gene editingcomponents (single guide RNA and Cas9 nuclease). The modificationsinclude targeted disruption of the T cell receptor (TCR) alpha constant(TRAC) and beta-2 microglobulin (B2M) loci, and the insertion of ananti-CD19 CAR transgene into the TRAC locus via an adeno-associatedvirus expression cassette. The anti-CD19 CAR (SEQ ID NO: 40) is composedof an anti-CD19 single-chain variable fragment comprising the SEQ ID NO:47, the CD8 transmembrane domain of SEQ ID NO: 32, a CD28 co-stimulatorydomain of SEQ ID NO: 36, and a CD3ζ signaling domain of SEQ ID NO: 38.

In this study, eligible human patients received an intravenous (IV)infusion of CTX110 following lymphodepleting (LD) chemotherapy.

Study Population

Dose escalation and cohort expansion include adult subjects with B cellmalignancies. Subjects are assigned to independent dose escalationgroups based on disease histology. Enrolled adult subjects include thosewith select subtypes of non-Hodgkin lymphoma (NHL), including diffuselarge B cell lymphoma (DLBCL) not otherwise specified (NOS), high gradeB cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements,transformed follicular lymphoma (FL), grade 3b FL or Richter'stransformation of CLL.

Study Purpose and Rationale

The purpose of the Phase 1 dose escalation study is to evaluate thesafety and efficacy of anti-CD19 allogeneic CRISPR-Cas9 engineered Tcells (CTX110 cells) in subjects with relapsed or refractory B cellmalignancies.

Outcomes for patients with relapsed/refractory B cell malignancies arehistorically poor. However, the use of autologous CAR T cell therapy inthis setting has produced complete and durable responses where previoustreatment options were palliative (June et al., (2018) Science, 359,1361-1365; Maus and June, (2016) Clin Cancer Res, 22, 1875-1884; Neelapuet al., (2017) N Engl J Med, 377,2531-2544; Schuster et al., (2019) NEngl J Med, 380, 45-56; Schuster et al., (2017) N Engl J Med, 377,2545-2554). Autologous CAR T cell therapies require patient-specificcell collection and manufacturing. Unfortunately, some patients are notcandidates to undergo leukapheresis, or they experience diseaseprogression or death while awaiting treatment. An allogeneicoff-the-shelf CAR T cell product such as CTX110 could provide thebenefit of immediate availability, reduce manufacturing variability, andprevent individual subject manufacturing failures.

Further, patients treated with multiple rounds of chemotherapy may haveT cells with exhausted or senescent phenotypes. The low response ratesin patients with chronic lymphocytic leukemia (CLL) treated withautologous CAR T cell therapy have been partially attributed to theexhausted T cell phenotype (Fraietta et al., (2018) Nat Med, 24,563-571; Riches et al., (2013) Blood, 121, 1612-1621). By starting withchemotherapy-naïve T cells from a healthy donor, allogeneic approachescould increase the consistency and potency of CAR T therapy as comparedto autologous products.

The main barrier to the use of allogeneic CAR T cells has been the riskof graft versus host disease (GvHD). CRISPR Cas9 gene-editing technologyallows for reliable multiplex cellular editing. The CTX110 manufacturingprocess couples the introduction of the CAR construct to the disruptionof the TRAC locus through homologous recombination. The delivery andprecise insertion of the CAR at the TRAC genomic locus using anAAV-delivered DNA donor template and HDR contrasts with the randominsertion of genetic material using lentiviral and retroviraltransduction methods. CAR gene insertion at the TRAC locus results inelimination of TCR in nearly all cells expressing the CAR, whichminimizes risk of GvHD. Furthermore, manufacturing from healthy donorcells removes the risk of unintentionally transducing malignant B cells(Ruella et al., (2018) Nat Med, 24, 1499-1503). This first-in-humantrial in subjects with relapsed/refractory B cell malignancies aims toevaluate the safety as well as efficacy of CTX110 with thisCRISPR-Cas9-modified allogeneic CAR T cell approach.

CTX110, a CD19-directed genetically modified allogeneic T-cellimmunotherapy, is manufactured from the cells of healthy donors;therefore, the resultant manufactured cells are intended to provide eachsubject with a consistent, final product of reliable quality.Furthermore, the manufacturing of CTX110, through precise delivery andinsertion of the CAR at the TRAC site using AAV and homology-directedrepair (HDR), does not present the risks associated with randominsertion of lentiviral and retroviral vectors.

Objectives

Primary objective, Part A (Dose escalation): To assess the safety ofescalating doses of CTX110 in combination with various lymphodepletionagents in subjects with relapsed or refractory B cell malignancies todetermine the recommended Part B dose.

Primary objective, Part B (Cohort expansion): To assess the efficacy ofCTX110 in subjects with relapsed or refractory B cell malignancies, asmeasured by objective response rate (ORR).

Secondary objectives (Parts A and B): To further characterize theefficacy, safety, and pharmacokinetics of CTX110.

Exploratory objectives (Parts A and B): To identify genomic, metabolic,and/or proteomic biomarkers associated with CTX110 that may indicate orpredict clinical response, resistance, safety, or pharmacodynamicactivity.

Endpoints Primary Endpoints

-   -   Part A: The incidence of adverse events, defined as        dose-limiting toxicities.    -   Part B: The objective response rate (ORR) defined as complete        response (CR)+partial response (PR) per the Lugano Response        Criteria for Malignant Lymphoma (Cheson et al., (2014) J Clin        Oncol, 32, 3059-3068), as determined by independent central        radiology review.

The Lugano Classification provides a standardized way to assess imagingin lymphoma subjects. It is comprised of radiologic assessments of tumorburden on diagnostic CT, and metabolic assessments on F¹⁸ FDG-PET forFDG-avid histologies (see Tables 13-14).

TABLE 13 Lugano Classification Assessment Components. Diagnostic CT/MRIF¹⁸ FDC-PET Target Lymph Nodes and Extra Nodal 5 Point Scale (Deauville)PET Score Lesions (Lymph Nodes and Extra Lymphatic Sites) * Up to 6 ofthe largest target nodes, nodal masses, The 5-point scale scores thesite of the most or other lymphomatous lesions that are intense FDGuptake for the time point, as follows: measurable in two diameters(longest diameter Score Criteria [LDi] and shortest diameter) should beidentified 1 No uptake from different body regions representative of the2 Uptake ≤ mediastinum subject's overall disease burden and include 3Uptake > mediastinum but ≤ liver mediastinal and retroperitonealdisease, if 4 Uptake moderately higher than liver involved. (moderatelyindicates uptake greater Nodal disease: Must have an LDi > 1.5 cm thannormal liver) Extranodal disease: Must have an 5 Uptake markedly higherthan liver LDi > 1.0 cm (markedly indicates much higher thanNon-Measured Lesions normal liver) All other lesions (including nodal,extranodal, and/or and assessable disease) should be followed as Newlesions nonmeasured disease (e.g., cutaneous, GI, bone, X New areas ofuptake unlikely to be spleen, liver, kidneys, pleural or pericardialrelated to lymphoma effusions, ascites). Bone Marrow: FDG uptakeassessed as Organ Enlargement (Spleen) No FDG uptake consistent withlymphoma The spleen is considered enlarged Focal FDG uptake consistentwith (splenomegaly) when >13 cm in the cranial to lymphoma caudaldimension. Diffuse FDG uptake consistent with New Lesions lymphoma Nodaldisease: Must have an LDi > 1.5 cm Extranodal disease: Any size CT:computed tomography; F¹⁸ FDG: fluorodeoxyglucose F18; LDi: longestdiameter; MRI: magnetic resonance imaging; PET: positron emissiontomography. * See (Barrington et al., (2014) J Clin Oncol, 32,3048-3058).

TABLE 14 Lugano Criteria for Response Assessment. At each follow-up timepoint, a PET-based response and a CT-based response is made per thedefinitions below. Response and Site PET-based Response CT-basedResponse COMPLETE Complete Metabolic Response* Complete RadiologicResponse ALL of the following ALL of the following Lymph nodes, Score of1, 2, or 3* Lymph nodes: All <1.5 cm in longest extranodal lesionsdiameter. Extralymphatic disease absent. Nonmeasured lesion N/A AbsentOrgan enlargement N/A Spleen: normal size New lesions No newmetabolically active lesions None (new lesions drive score 5) Bonemarrow No FDG-avid disease in marrow Normal by morphology; ifindeterminate, IHC negative. PARTIAL Partial Remission Partial MetabolicResponse ALL of the following Lymph nodes, Score of 4, or 5 with reduceduptake ≥50% decrease in SPD of all extranodal lesions from baseline andresidual masses of target lesions from baseline any size Nonmeasuredlesion N/A Absent, normal, or regressed, but no increase Organenlargement N/A Spleen: ≥50% decrease from baseline in enlarged portionNew lesions None None Bone marrow Residual uptake higher than uptake inN/A normal marrow but reduced compared with baseline (e.g., persistentfocal changes in the marrow with nodal response) NO RESPONSE/STABLEDISEASE No Metabolic Response Stable Disease Lymph nodes, Score of 4, or5 with no significant <50% decrease in SPD of all target extranodallesions change in FDG uptake from baseline lesion from baseline Noprogression Nonmeasured lesion N/A No increase consistent withprogression Organ enlargement N/A Spleen: No increase consistent withprogression New lesions None None Bone marrow No change from baselineN/A PROGRESSION Progressive Disease Progressive Metabolic Response ANYof the following Lymph nodes, Lymph nodes/nodal masses: PPD Progressionextranodal lesions Score of 4 or 5 with increased An individualnode/extranodal lesion must uptake compared to baseline. be abnormal(nodal disease with LDi > 1.5 Extranodal lesions: New FDG cm, extranodaldisease with and LDi > 1.0 avid foci consistent with cm) with: lymphoma.Increase of ≥50% from the product of the perpendicular diameters (PPD)from nadir AND Increase in LDi or SDi from nadir ≥0.5 cm for lesions ≤2cm ≥1.0 cm for lesions >2 cm Nonmeasured lesion None Unequivocalprogression Organ enlargement None Progression of pre-existingsplenomegaly: Splenic length must increase by 50% of the extent of itsprior increase beyond baseline (e.g., a 15-cm spleen must increase to 16cm). New splenomegaly: Spleen must increase by at least 2 cm frombaseline Or Recurrent splenomegaly New lesions New FDG-avid fociconsistent with Regrowth of previously resolved lesions lymphoma ratherthan another etiology New node > 1.5 cm in any axis New extranodalsite > 1.0 cm in any axis New extranodal site < 1.0 cm in any axis orunequivocal/attributable to lymphoma New assessable disease unequivocal/attributable to lymphoma of any size Bone marrow New/recurrent FDG-avidfoci New or recurrent involvement FDG: fluorodeoxyglucose; NC:immunohistochemistry; LDi: longest diameter; N/A: not applicable; PPD:perpendicular diameters; SDi: shortest diameter; SPD: sum of theproducts of diameters. *Deauville score of 3 represent a completemetabolic response (Barrington et al., (2014) J Clin Oncol, 32,3048-3058). Note: It is recognized that in Waldeyer's ring or extranodalsites with high physiologic uptake or with activation within spleen ormarrow (e.g., with chemotherapy or myeloid colony-stimulating factors),uptake may be greater than normal mediastinum and/or liver. In thiscircumstance, complete metabolic response may be inferred if uptake atsites of initial involvement is no greater than surrounding normaltissue even if the tissue has high physiologic uptake.

Secondary Endpoints (Dose Escalation and Cohort Expansion)

Efficacy

-   -   Duration of response/remission (central read/assessment).        Duration of response/remission is reported only for subjects who        have had objective response events. This is calculated as the        time between first objective response and date of disease        progression or death due to any cause.    -   Progression-free/event-free survival (central read/assessment).        Progression-free survival (PFS) and event-free survival is        calculated as the difference between date of CTX110 infusion and        date of disease progression or death due to any cause. Subjects        who have not progressed and are still on study at the data        cutoff date are censored at their last assessment date.    -   Overall survival. Overall survival is calculated as the time        between date of first dose of CTX110 and death due to any cause.        Subjects who are alive at the data cutoff date are censored at        their last date known to be alive.

Safety

Frequency and severity of AEs and clinically significant laboratoryabnormalities.

Pharmacokinetic

-   -   Levels of CTX110 in blood over time.

Exploratory Endpoints (Dose Escalation and Cohort Expansion)

-   -   Levels of CTX110 in tissues (e.g., trafficking of CTX110 in bone        marrow, CSF, and/or tumor tissue may be evaluated in any samples        collected per protocol-specific sampling).    -   Levels of cytokines in blood and other tissues.    -   Incidence of anti-CTX110 antibodies.    -   Levels of B cells and immunoglobulins overtime.    -   Impact of anti-cytokine therapy on CTX110 proliferation, CRS,        and response.    -   Incidence of autologous or allogeneic HSCT following CTX110        therapy.    -   Incidence and type of subsequent anticancer therapy.    -   Time to complete response/remission.    -   First subsequent therapy-free survival.    -   Other genomic, protein, metabolic, or pharmacodynamic endpoints.

Study Design

This is an open-label, multicenter, Phase 1 study evaluating the safetyand efficacy of CTX110 in subjects with relapsed or refractory B cellmalignancies. The study is divided into 2 parts: dose escalation (PartA) followed by cohort expansion (Part B).

Part A investigates escalating doses of CTX110 in Cohort A in adultsubjects with 1 of the following NHL subtypes: DLBCL NOS, high grade Bcell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, grade 3b FL,or transformed FL.

In the dose escalation part of the study, 1 additional cohort (Cohort B)with an NHL population similar to Cohort A has been added to explore anincreased dose of cyclophosphamide (750 mg/m²) relative to Cohort A (500mg/m²). Subjects in Cohort B are treated with an increased dose ofcyclophosphamide to explore the effects of a longer suppression oflymphocytes on CAR T cell expansion following CTX110 infusion (see Table15).

TABLE 15 Cohort A and Cohort B. Cohort Disease Subset Treatment A Adultsubjects with DLBCL NOS, LD chemotherapy: Co-administration of highgrade B cell lymphoma with fludarabine MYC and BCL2 and/or BCL6 30mg/m² + cyclophosphamide 500 mg/m² IV rearrangements, grade 3b FL, anddaily for 3 days transformed FL CTX110 starting at DL1 B Same as CohortA LD chemotherapy: Co-administration of fludarabine 30 mg/m² +cyclophosphamide 750 mg/m² IV daily for 3 days CTX110 starting at DL2DL1/2: Dose Level 1 or 2; DLBCL: diffuse large B cell lymphoma; FL:follicular lymphoma; IV: intravenously; LD: lymphodepleting.

The study is divided into 2 parts: dose escalation (Part A) followed bycohort expansion (Part B). Both parts of the study will consist of 3main stages: screening, treatment, and follow-up. A schematic depictionof the study schema is shown in FIG. 21.

A schedule of assessments is provided in Table 16 and Table 17.

Stage 1—Screening to determine eligibility for treatment (up to 14days).Stage 2—Lymphodepleting (LD) chemotherapy and infusion of CTX110 (1-2weeks). Prior to both the initiation of LD chemotherapy and infusion ofCTX110, the clinical eligibility of subjects must be reconfirmed.

Stage 2A—LD chemotherapy:

-   -   Cohort A: Co-administration of fludarabine 30 mg/m² and        cyclophosphamide 500 mg/m² intravenously (IV) daily for 3 days.    -   Cohort B: Co-administration of fludarabine 30 mg/m² and        cyclophosphamide 750 mg/m² intravenously (IV) daily for 3 days.

Stage 2B—CTX110 infusion:

-   -   Cohort A (NHL subsets): Lymphodepleting (LD) chemotherapy        (fludarabine 30 mg/m² and cyclophosphamide 500 mg/m²        intravenously [IV] daily for 3 days) completed at least 48 hours        (but no more than 7 days) prior to CTX110 infusion (dose        escalation from Dose Level [DL] 1).    -   Cohort B (higher LD chemotherapy dose): LD chemotherapy        (fludarabine 30 mg/m² and cyclophosphamide 750 mg/m² IV daily        for 3 days) completed at least 48 hours (but no more than 7        days) prior to CTX110 infusion (dose escalation from Dose Level        [DL] 2).

Stage 3—Follow up (5 years after the last CTX110 infusion).

For both dose escalation and cohort expansion, subjects must remainwithin proximity of the investigative site (i.e., 1-hour transit time)for 28 days after CTX110 infusion. During this acute toxicity monitoringperiod, subjects will be routinely assessed for adverse events (AEs),including cytokine release syndrome (CRS), neurotoxicity, and GvHD.Toxicity management guidelines are provided in the study protocol.During dose escalation, all subjects will be hospitalized for the first7 days following CTX110 infusion, or longer if required by localregulation or site practice.

After the acute toxicity monitoring period, subjects will besubsequently followed for up to 5 years after CTX110 infusion withphysical exams, regular laboratory and imaging assessments, and AEevaluations. After completion of this study, subjects will be requiredto participate in a separate long-term follow-up study for an additional10 years to assess long-term safety and survival.

LD chemotherapy it to be delayed if any of the following signs orsymptoms are present:

-   -   Significant worsening of clinical status that, according to the        investigator, increases the potential risk of AEs associated        with LD chemotherapy.    -   Requirement for supplemental oxygen to maintain a saturation        level of >91%.    -   New uncontrolled cardiac arrhythmia.    -   Hypotension requiring vasopressor support.    -   Active infection: Positive blood cultures for bacteria, fungus,        or virus not responding to treatment.    -   Grade ≥2 acute neurological toxicity.

TABLE 16 Schedule of Assessments (Screening to Month 24). Treat- mentScreen- (Stage 2) Study ing ¹ D-5 Follow-up (Stage 3) Stage (Stage to D2± D3 ± D5 ± D8 ± D10 ± D14 ± D21 ± Day 1) D-3 D1² 2 d 2 d 2 d 2 d 2 d 2d 2 d Informed X consent Medical X history³ Physical X X X X X X X X X Xexam Vital X X X X X X X X X X signs ⁴ Height, X X X X X weight ⁵ Preg-X X nancy test ⁶ ECOG X X status Echo- X cardio- gram 12-lead X X X ECG⁷ Brain X MRI Lumbar X punc- ture ⁸ ICE X X X X X X assess- ment ⁹Patient- X reported outcome Con- Continuous comitant meds ¹⁰ AdverseContinuous events ¹¹ Hospital Continuous util- ization Treat- ment LD Xchemo- ther- apy ¹² CTX110 X infu- sion ¹³ NHL Disease Re- sponse/Assess- ment (Central and Local) PET/CT X scan ¹⁴ BM X biopsy ¹⁵ Tumorbiopsy ¹⁶ Tumor X path- ology¹⁷ Adult B Cell ALL Disease Re- sponse/Assess- ment BM X biopsy and aspirate (central and lo- cal) ^(14, 15)Peri- X pheral blood chim- erism (local) ¹⁹ Lab - oratory Assess- ments(Local) CBC w/ X X X X X X X X X X differ- ential Serum X X X X X X X XX X chemistry Coagu- X X X X X X X X X lation para- meters Viral Xserol- ogy ²⁰ Immuno- X X globulins Ferritin, X X X X X X X X X CRPLympho- X X X X X X X X cyte subsets ²¹ B cells X X X (CD19, CD20) Bloodtype, Ab screen ²² Bio- markers (Blood, Central) CTX110 X X25 X X X X XX X PK ^(23, 24) pre/ post Cyto- X X X X X X X X X kines ²⁶ Anti- X Cas9Ab ²⁴ Anti- X CTX110 Ab ²⁴ Immuno- X X25 X X X X X pheno- pre/ type postDNA X Cell-free X DNA PBMCs X Explor- X X²⁸ X X X X X X X X atory bio-mar- kers ²⁷ Study Follow-up (Stage 3) Stage D28 ± M2 ± M3 ± M6 ± M9 ±M12 ± M15 ± M18 ± M24 ± Day 4 d 7 d 7 d 14 d 14 d 14 d 14 d 14 d 21 dInformed consent Medical history³ Physical X X X X X X X X X exam VitalX X X X X X X X X signs ⁴ Height, X X X X X X X X X weight ⁵ Preg- nancytest ⁶ ECOG X X status Echo- cardio- gram 12-lead X ECG ⁷ Brain MRILumbar punc- ture ⁸ ICE X assess- ment ⁹ Patient- X X X X reportedoutcome Con- Continuous comitant meds ¹⁰ Adverse Continuous events ¹¹Hospital Continuous util- ization Treat- ment LD chemo- ther- apy ¹²CTX110 infu- sion ¹³ NHL Disease Re- sponse/ Assess- ment (Central andLocal) PET/CT X X X X X X X scan ¹⁴ BM X biopsy ¹⁵ Tumor X biopsy ¹⁶Tumor path- ology¹⁷ Adult B Cell ALL Disease Re- sponse/ Assess- ment BMX X¹⁸ X¹⁸ biopsy and aspirate (central and lo- cal) ^(14, 15) Peri-pheral blood chim- erism (local) ¹⁹ Lab - oratory Assess- ments (Local)CBC w/ X X X X X X X X X differ- ential Serum X X X X X X X X Xchemistry Coagu- X lation para- meters Viral serol- ogy ²⁰ Immuno- X X XX X X X X X globulins Ferritin, X CRP Lympho- X X X X X X cyte subsets²¹ B cells X X X X X X X X X (CD19, CD20) Blood type, Ab screen ²² Bio-markers (Blood, Central) CTX110 X X X X X X X X X PK ^(23, 24) Cyto- X Xkines ²⁶ Anti- X X X X Cas9 Ab ²⁴ Anti- X X X X CTX110 Ab ²⁴ Immuno- X XX X X X X X X pheno- type DNA Cell-free X X X X X X X DNA PBMCs X X X XX X X Explor- X X X X X X X X X atory bio- mar- kers ²⁷ Ab: antibody;AE: adverse event; BM: bone marrow; Cas9: CRISPR-associated protein 9;CBC: complete blood count; CNS: central nervous system; CRISPR:clustered regularly interspaced short palindromic repeats; CRP:C-reactive protein; CT: computed tomography; D or d: day; EC₉₀: 90%effectiveconcentration; ECG: electrocardiogram; ECOG: EasternCooperative Oncology Group; HBV: hepatitis B virus; HCV: hepatitis Cvirus; HIV-1/-2: human immunodeficiency virus type 1 or 2; HSCT:hematopoietic stem cell transplant; ICE: immune effector cell-associatedencephalopathy; ICF: informed consent form; IPI: InternationalPrognostic Index; LD: lymphodepleting; LP: lumbar puncture; M: month;MRI: magnetic resonance imaging; PBMC: peripheral blood mononuclearcell; PCR: polymerase chain reaction; PET: positron emission tomography;PK: pharmacokinetic(s); Q: every; TBNK: T-, B-, natural killer (cells).¹ Screening assessments completed within 14 days of informed consent.Subjects allowed 1-time rescreening within 3 months of initial consent.² All baseline assessments on Day 1 are to be performed prior to CTX110infusion unless otherwise specified. ³ Includes complete surgical andcardiac history. ⁴ Includes sitting blood pressure, heart rate,respiratory rate, pulse oximetry, and temperature. ⁵ Height at screeningonly. ⁶ For female subjects of childbearing potential. Serum pregnancytest at screening. Serum or urine pregnancy test within 72 hours beforestart of LD chemotherapy. ⁷ Prior to LD chemotherapy, and prior toCTX110 infusion. ⁸ LP at screening on subjects with high risk for CNSinvolvement (e.g., high-grade B cell lymphoma with MYC and BCL2 and/orBCL6 rearrangements, subjects with testicular involvement of lymphoma,or subjects with high-risk CNS IPI score). For LPs performed duringneurotoxicity, samples should be sent to central laboratory for CTX110PK and exploratory biomarkers whenever possible. ⁹ On Day 1 prior toCTX110 administration. If CNS symptoms persist after Day 28, ICEassessment should continue to be performed approximately every 2 daysuntil symptom resolution to grade 1 or baseline. ¹⁰ All concomitantmedications will be collected ≤ 3 months post-CTX110, after which onlyselect concomitant medications will be collected. 11 Collect all AEsfrom informed consent to Month 3 visit, collect all SAEs and AESIs afterMonth 3 visit to Month 60 visit. Only SAEs and AESIs should be reportedfor ≤ 3 months post-CTX110 if subject begins new anticancer therapybefore Month 3 visit. Only AESIs will be reported if subject begins newanticancer therapy after Month 3 visit. ¹² Start LD chemotherapy within7 days of study enrollment. After completion of LD chemotherapy, ensurewashout period of ≥ 48 hours (but ≤ 7 days) before CTX110 infusion.Physical exam, weight, and coagulation laboratories performed prior tofirst dose of LD chemotherapy. Vital signs, CBC, clinical chemistry, andAEs/concomitant medications assessed and recorded daily (i.e., 3 times)during LD chemotherapy. ¹³ CTX110 administered 48 hours to 7 days aftercompletion of LD chemotherapy. ¹⁴ Baseline disease assessment (PET/CTfor subjects with NHL) to be performed within 28 days prior to CTX110infusion. MRI with contrast allowed if CT clinically contraindicated, oras required by local regulation. ¹⁵ BM biopsy to confirm completeresponse as part of disease evaluation. BM biopsy may also be performedat time of disease relapse. Samples from BM aspirate after CTX110infusion should be sent for CTX110 PK and exploratory biomarkers. To beperformed ± 5 days of visit date. ¹⁶ Optional: For subjects who havedisease amenable to biopsy and who provide separate consent. To beperformed ± 5 days of visit date. ¹⁷It is preferred that subjectsundergo tumor biopsy during screening. However, if a biopsy ofrelapsed/refractory disease was performed within 3 months prior toenrollment and after the most recent line of therapy, archival tissuemay be used. If relapse occurs on study, every attempt should be made toobtain biopsy of relapsed tumor and send to central pathology. Tumorbiopsy refers to tissue other than bone marrow. ¹⁸ Assessments at Months2 and 3 to confirm CR if not achieved at Month 1. ¹⁹ To be performedonly in subjects who have received prior allogeneic HSCT. ²⁰ Infectiousdisease testing (HIV-1, HIV-2, HCV antibody/PCR, HBV surface antigen,HBV surface antibody, HBV core antibody) ≤ 30 days of signing ICF may beconsidered for subject eligibility. ²¹ Lymphocyte subset assessment atscreening, before start of first day of LD chemotherapy, before CTX110infusion, then all listed time points. To include 6-color TBNK panel, orequivalent for T, B, and natural killer cells. ²² Blood type andantibody screen. ²³ Samples for CTX110 PK should be sent from any LP, BMbiopsy, or tissue biopsy performed following CTX110 infusion. If CRSoccurs, samples for assessment of CTX110 levels will be collected every48 hours between scheduled visits until CRS resolves. ²⁴ Sponsor mayrequest discontinuation of sample collection if consecutive tests arenegative. Continue sample collection for all listed time points untilotherwise instructed by sponsor. ²⁵ Two samples collected on Day 1: Onepre-CTX110 infusion and one 20 (± 5) minutes after the end of CTX110infusion. ²⁶ Additional cytokine samples should be collected daily forthe duration of CRS. Day 1 samples to be collected prior to CTX110infusion. ²⁷ Samples for exploratory biomarkers should be sent from anyLP or BM biopsy performed following CTX110 infusion. If CRS occurs,samples for assessment of exploratory biomarkers will be collected every48 hours between scheduled visits until CRS resolves. ²⁸ Prior to firstday of LD chemotherapy only.

TABLE 17 Schedule of Assessments (Months 30-60). M30 (± M36 (± M42 (±M48(± M54(± M60 (± Progressive Secondary Assessments 21 days) 21 days)21 days) 21 days) 21 days) 21 days) Disease Follow-Up ¹ Vital signs ² XX X X X X X X Physical exam X X X X X X X X Concomitant medications ³ XX X X X X X X Disease assessment ⁴ X X X X X X X CBC with differential ⁵X X X X X X X X Serum chemistry ⁵ X X X X X X X X Immunoglobulins^(5, 6) X X X X X X X Lymphocyte subsets ^(5, 6) X X X X X X X CTX110persistence X X X X X X X X (blood, central) ^(6, 7) Exploratorybiomarkers X X X X X X X X (blood, central) Anti-Cas9 Ab X X X X (blood,central) ⁶ Anti-CTX110 , X X X X anti-daratumumab Ab (blood, central) ⁶Patient-reported outcome X X X X Adverse events ⁸ X X X X X X X X Ab:antibody; AESI: adverse event of special interest; BM: bone marrow;Cas9: CRISPR-associated protein 9; CBC: complete blood count; CRISPR:clustered regularly interspaced short palindromic repeats; CT: computedtomography; NHL: non-Hodgkin lymphoma; PET: positron emissiontomography; PK: pharmacokinetic; SAE: serious adverse event; TBNK: T-,B-, natural killer (cells). ¹ Subjects with progressive disease or whoundergo stem cell transplant will discontinue the normal schedule ofassessments and attend annual study visits. Visits will occur at12-month intervals. Subjects who partially withdraw consent will undergothese procedures at minimum. ² Includes temperature, blood pressure,pulse rate, and respiratory rate. ³ Only select concomitant medicationswill be collected. ⁴ Disease assessment will consist of investigatorreview of physical exam, CBC, clinical chemistry, and lactatedehydrogenase for NHL subjects, and of physical exam, CBC withdifferential, and clinical chemistry for B cell ALL. NHL subjects withsuspected malignancy will undergo PET/CT imaging and/or a BM biopsy toconfirm relapse. Every attempt should be made to obtain a biopsy of therelapsed tumor in subjects who progress. Assessed at local laboratory.To include 6-color TBNK panel, or equivalent for T, B, and naturalkiller cells. ⁶ Sponsor may request discontinuation of samplecollection. Continue sample collection for all listed time points untilotherwise instructed by sponsor. ⁷ Samples for CTX110 PK analysis shouldbe sent to the central laboratory from any lumbar puncture, BM biopsy,or tissue biopsy performed following CTX110 infusion. ⁸ SAEs and AESIsshould be reported for up to 3 months after CTX110 infusion if a subjectbegins new anticancer therapy before Month 3 study visit. Only AESIswill be reported if a subject begins new anticancer therapy after Month3 study visit.

The goal of lymphodepletion is to allow for significant CAR T cellexpansion following infusion. LD chemotherapy consisting of fludarabineand cyclophosphamide across different doses has been successfullyutilized in several autologous CAR T cell trials. The rationale for theuse of LD chemotherapy is to eliminate regulatory T cells and othercompeting elements of the immune system that act as ‘cytokine sinks,’enhancing the availability of cytokines such as interleukin 7 (IL-7) andinterleukin 15 (IL-15) (Dummer et al., (2002) J Clin Invest, 110,185-192; Gattinoni et al., (2005) J Exp Med, 202, 907-912).Additionally, it is postulated that naïve T cells begin to proliferateand differentiate into memory-like T cells when total numbers of naïve Tcells are reduced below a certain threshold (Dummer et al., (2002) JClin Invest, 110, 185-192). Cohort A will use cyclophosphamide (500mg/m²) and fludarabine (30 mg/m²) at doses that are consistent withdoses used in registrational clinical trials of axicabtagene ciloleucel.Cohort B will use a higher dose of cyclophosphamide (750 mg/m²) toevaluate whether increased intensity of lymphodepletion may facilitateexpansion of an allogeneic CAR T cell product. Doses of cyclophosphamidewithin this range (total of >120 mg/kg or 3 g/m²) have been used inprior CAR T cell therapy studies in hematological malignancies(Brentjens et al., (2011) Blood, 118, 4817-4828; Kochenderfer et al.,(2015) J Clin Oncol, 33, 540-549; Turtle et al., (2016) Sci Transl Med,8, 355ra116). When used as a part of higher intensity LD chemotherapy,increased doses of cyclophosphamide are associated with improvedefficacy (Hirayama et al., (2019) Blood, 133, 1876-1887).

CTX110 infusion is to be delayed if any of the following signs orsymptoms are present:

-   -   New active uncontrolled infection.    -   Worsening of clinical status compared to prior to start of LD        chemotherapy that, in the opinion of the investigator, places        the subject at increased risk of toxicity.    -   Grade ≥2 acute neurological toxicity.

CTX110 Dose

CTX110 cells are administered IV using a flat dosing schema based on thenumber of CAR+ T cells. The starting dose is 3×10⁷ CAR+ T cells, whichis approximately 1 log lower than the doses of autologous CAR T cellscurrently approved for NHL including KYMRIAH® (5×10⁸ total CAR T cells)and YESCARTA® (2×10⁶ kg, maximum 2×10⁸ CAR T cells).

Dose Escalation

Dose escalation will be performed using a standard 3+3 design. Thefollowing doses of CTX110, based on CAR⁺ T cells, may be evaluated inthis study beginning with DL1 for Cohort A. Only after assessment andconfirmation of safety of DL2 in Cohort A by the Safety Review Committee(SRC) may subsequent Cohort B be opened/enrolled and begin doseescalation from DL2. Due to the study's dose limit of 7×10⁴ TCR+cells/kg, the study may proceed with DL4 in Cohort A and/or Cohort B ifa subject weighs ≥60 kg (see Table 18).

TABLE 18 Dose Levels. Dose Level Total CAR+ T Cell Dose −1 1 × 10⁷ 1 3 ×10⁷ 2 1 × 10⁸ 3 3 × 10⁸ 4 1 × 10⁹ CAR: chimeric antigen receptor.

The DLT evaluation period begins with CTX110 infusion and last for 28days. The first 3 subjects in each cohort will be treated in a staggeredmanner, such that the 2^(nd) and 3^(rd) subjects will only receiveCTX110 after the previous subject has completed the DLT evaluationperiod. In subsequent dose levels or expansion of the same dose level,cohorts of up to 3 subjects may be enrolled and dosed concurrently.

Subjects must receive CTX110 to be evaluated for DLT. If a subjectdiscontinues the study any time prior to CTX110 infusion, the subjectwill not be evaluated for DLT and a replacement subject will be enrolledat the same dose level as the discontinued subject. If a DLT-evaluablesubject has signs or symptoms of a potential DLT, the DLT evaluationperiod will be extended according to the protocol-defined window toallow for improvement or resolution before a DLT is declared.

Toxicities are graded and documented according to National CancerInstitute Common Terminology Criteria for Adverse Events (CTCAE) version5, except for CRS (Lee criteria), neurotoxicity (ICANS, immune effectorcell-associated neurotoxicity syndrome criteria and CTCAE v5.0), andGvHD (Mount Sinai Acute GVHD International Consortium [MAGIC] criteria).

A DLT will be defined as any of the following events occurring duringthe DLT evaluation period that persists beyond the specified duration(relative to the time of onset):

-   -   Grade ≥2 GvHD that is steroid-refractory (e.g., progressive        disease after 3 days of steroid treatment [e.g., 1 mg/kg/day],        stable disease after 7 days, or partial response after 14 days        of treatment).    -   Death during the DLT period (except due to disease progression).    -   Any grade 3 or 4 toxicity that is clinically significant        according to the investigator's judgement and does not improve        within 72 hours.    -   The following will NOT be considered as DLTs:        -   Grade 3 or 4 CRS that improves to grade ≤2 within 72 hours.        -   Grade 3 or 4 neurotoxicity (e.g., encephalopathy, confusion)            that improves to grade ≤2 within 14 days.        -   Grade 3 or 4 fever.        -   Bleeding in the setting of thrombocytopenia (platelet count            <50×10⁹/L); documented bacterial infections or fever in the            setting of neutropenia (absolute neutrophil count            <1000/mm³).        -   Grade 3 or 4 hypogammaglobulinemia.        -   Grade 3 or 4 pulmonary toxicity that resolves to grade ≤2            within 7 days. For subjects intubated due to fluid overload            from supportive care, this may be extended to 14 days.        -   Grade 3 or 4 liver function studies that improve to grade ≤2            within 14 days.        -   Grade 3 or 4 renal insufficiency that improves to grade ≤2            within 21 days.        -   Grade 3 or 4 thrombocytopenia or neutropenia will be            assessed retrospectively. After at least 6 subjects are            infused, if ≥50% of subjects have prolonged cytopenias            (i.e., lasting more than 28 days post-infusion), dose            escalation will be suspended pending SRC assessment.

AEs that have no plausible causal relationship with CTX110 will not beconsidered DLTs.

Toxicity Management

Subjects must be closely monitored for at least 28 days after CTX110infusion. Significant toxicities have been reported with autologous CART cell therapies and investigators are required to proactively monitorand treat all adverse events in accordance with protocol guidance.

The following general recommendations are provided based on priorexperience with CD19-directed autologous CAR T cell therapies:

-   -   Fever is the most common early manifestation of cytokine release        syndrome (CRS); however, subjects may also experience weakness,        hypotension, or confusion as first presentation.    -   Diagnosis of CRS should be based on clinical symptoms and NOT        laboratory values.    -   In subjects who do not respond to CRS-specific management,        always consider sepsis and resistant infections. Subjects should        be continually evaluated for resistant or emergent bacterial        infections, as well as fungal or viral infections.    -   CRS, hemophagocytic lymphohistiocytosis (HLH), and tumor lysis        syndrome (TLS) may occur at the same time following CAR T cell        infusion. Subjects should be consistently monitored for signs        and symptoms of all the conditions and managed appropriately.    -   Neurotoxicity may occur at the time of CRS, during CRS        resolution, or following resolution of CRS. Grading and        management of neurotoxicity will be performed separately from        CRS.    -   Tocilizumab must be administered within 2 hours from the time of        order.

The safety profile of CTX110 will be continually assessed throughout thestudy, and investigators will be updated on a regular basis with newinformation regarding the identification and management of potentialCTX110-related toxicity.

Infusion Reactions

Infusion reactions have been reported in autologous CD19-directed CAR Tcell trials, including transient fever, chills, and/or nausea.Acetaminophen (paracetamol) and diphenhydramine hydrochloride (oranother H1-antihistamine) may be repeated every 6 hours after CTX110infusion, as needed, if an infusion reaction occurs. Nonsteroidalanti-inflammatory medications may be prescribed, as needed, if thesubject continues to have fever not relieved by acetaminophen. Systemicsteroids should not be administered except in cases of life-threateningemergency, as this intervention may have a deleterious effect on CAR Tcells.

Febrile Reaction and Infection Prophylaxis

Infection prophylaxis should occur according to the institutionalstandard of care for patients with B cell malignancies in animmunocompromised setting. In the event of febrile reaction, anevaluation for infection should be initiated and the subject managedappropriately with antibiotics, fluids, and other supportive care asmedically indicated and determined by the treating physician. Viral andfungal infections should be considered throughout a subject's medicalmanagement if fever persists. If a subject develops sepsis or systemicbacteremia following CTX110 infusion, appropriate cultures and medicalmanagement should be initiated. Additionally, consideration of CRSshould be given in any instances of fever following CTX110 infusionwithin 30 days post-infusion.

Tumor Lysis Syndrome (TLS)

Subjects receiving CAR T cell therapy are at increased risk of TLS.Subjects should be closely monitored for TLS via laboratory assessmentsand symptoms from the start of LD chemotherapy until 28 days followingCTX110 infusion. All subjects should receive prophylactic allopurinol(or a non-allopurinol alternative, such as febuxostat) and increasedoral/IV hydration during screening and before initiation of LDchemotherapy. Prophylaxis can be stopped after 28 days following CTX110infusion or once the risk of TLS passes.

Sites should monitor and treat TLS as per their institutional standardof care, or according to published guidelines (Cairo and Bishop, (2004)Br J Haematol, 127, 3-11). TLS management, including administration ofrasburicase, should be instituted promptly when clinically indicated.

Cytokine Release Syndrome (CRS)

CRS is a major toxicity reported with autologous CD19-directed CAR Tcell therapy. CRS is due to hyperactivation of the immune system inresponse to CAR engagement of the target antigen, resulting inmulti-cytokine elevation from rapid T cell stimulation and proliferation(Frey et al., (2014) Blood, 124, 2296; Maude et al., (2014) Cancer J,20, 119-122). When cytokines are released, a variety of clinical signsand symptoms associated with CRS may occur, including cardiac,gastrointestinal (GI), neurological, respiratory (dyspnea, hypoxia),skin, cardiovascular (hypotension, tachycardia), and constitutional(fever, rigors, sweating, anorexia, headaches, malaise, fatigue,arthralgia, nausea, and vomiting) symptoms, and laboratory (coagulation,renal, and hepatic) abnormalities.

The goal of CRS management is to prevent life-threatening sequelae whilepreserving the potential for the antitumor effects of CTX110. Symptomsusually occur 1 to 14 days after autologous CAR T cell therapy, but thetiming of symptom onset has not been fully defined for allogeneic CAR Tcells.

CRS should be identified and treated based on clinical presentation andnot laboratory cytokine measurements. If CRS is suspected, grading andmanagement should be performed according to the recommendations in Table19, which are adapted from published guidelines (Lee et al., (2014)Blood, 124, 188-195). Since the development of the original Lee CRSgrading criteria, physicians using CAR T cell therapies have gainedfurther understanding of the presentation and time course of CRS. Therecent American Society for Blood and Marrow Transplantation (ASBMT)consensus criteria (Lee et al., (2018) Biol Blood Marrow Transplant)recommend that grading should be based on the presence of fever withhypotension and/or hypoxia, and that other end organ toxicities shouldbe managed separately with supportive care. Accordingly, in thisprotocol neurotoxicity will be graded and managed using a differentscale (see section entitled “Immune Effector Cell-AssociatedNeurotoxicity Syndrome (ICANS)”), and end organ toxicity in the contextof CRS management refers only to hepatic and renal systems (as in thePenn Grading criteria; (Porter et al., (2018) J Hematol Oncol, 11, 35).The sponsor may elect to revise the CRS grading criteria and toxicitymanagement algorithms to reflect the ASBMT consensus proposal based onclinical experience with CTX110 and other CAR T cell therapies.

TABLE 19 Cytokine Release Syndrome Grading and Management Guidance. CRSSeverity ¹ Tocilizumab Corticosteroids Grade 1 N/A N/A Symptoms requiresymptomatic treatment only (e.g., fever, fatigue, headache, myalgia,malaise). Grade 2 Administer tocilizumab³ 8 mg/kg Manage per grade 3 ifno Symptoms require and respond to IV over 1 hour (not to exceedimprovement within 24 hours moderate intervention. Oxygen 800 mg). afterstarting tocilizumab. requirement < 40% FiO₂ or Repeat tocilizumab every8 hypotension responsive to fluids hours as needed if not responsive orlow dose of 1 vasopressor or to IV fluids or increasing grade 2 organtoxicity.² supplemental oxygen. Limit to ≤3 doses in a 24-hour period;maximum total of 4 doses. Grade 3 Per grade 2. If no improvement within24 Symptoms require and respond to hours after starting tocilizumab,aggressive intervention. Oxygen administer methylprednisolonerequirement ≥40% FiO₂ or 1 mg/kg IV twice daily. hypotension requiringhigh-dose Continue corticosteroid use until or multiple vasopressors⁴ orgrade the event is grade ≤1, then taper 3 organ toxicity or grade 4 over3 days. transaminitis Grade 4 Per grade 2. Per grade 3. Life-threateningsymptoms. If no response to multiple doses Requirements for ventilatorof tocilizumab and steroids, support, continuous veno-venous considerusing other anti- hemodialysis or grade 4 organ cytokine therapies(e.g., toxicity (excluding transaminitis) siltuximab). CRS: cytokinerelease syndrome; FiO₂: fraction of inspired oxygen; IV: intravenously;N/A: not applicable. ¹ See (Lee et al., (2014) Blood, 124, 188-195).²Refer to entitled “Immune Effector Cell-Associated NeurotoxicitySyndrome (ICANS)” for management of neurologic toxicity. Organ toxicityrefers to hepatic and renal systems only. ³Refer to tocilizumabprescribing information. ⁴ See Table 20 for information on high-dosevasopressors.

TABLE 20 High-dose Vasopressors. Pressor Dose* Norepinephrinemonotherapy ≥20 μg/min Dopamine monotherapy ≥10 μg/kg/min Phenylephrinemonotherapy ≥200 μg/min Epinephrine monotherapy ≥10 μg/min If onvasopressin Vasopressin + norepinephrine equivalent of ≥10 μg/min** Ifon combination vasopressors Norepinephrine equivalent (not vasopressin)of ≥20 μg/min** *All doses are required for ≥3 hours. **VASST Trialvasopressor equivalent equation: norepinephrine equivalent dose =[norepinephrine (μg/min) + (μg/min)/2] + [epinephrine (μg/min)] +[phenylephrine (μg/min)/10]

Throughout the duration of CRS, subjects should be provided withsupportive care consisting of antipyretics, IV fluids, and oxygen.Subjects who experience grade ≥2 CRS (e.g., hypotension, not responsiveto fluids, or hypoxia requiring supplemental oxygenation) should bemonitored with continuous cardiac telemetry and pulse oximetry. Forsubjects experiencing grade 3 CRS, consider performing an echocardiogramto assess cardiac function. For grade 3 or 4 CRS, consider intensivecare supportive therapy. Intubation for airway protection due toneurotoxicity (e.g., seizure) and not due to hypoxia should not becaptured as grade 4 CRS. Similarly, prolonged intubation due toneurotoxicity without other signs of CRS (e.g., hypoxia) is notconsidered grade 4 CRS. Investigators should always consider thepotential of an underlying infection in cases of severe CRS, as thepresentation (fever, hypotension, hypoxia) is similar. Resolution of CRSis defined as resolution of fever (temperature ≥38° C.), hypoxia, andhypotension (Lee et al., (2018) Biol Blood Marrow Transplant).

Immune Effector Cell-associated Neurotoxicity Syndrome (ICANS)Neurotoxicity has been observed with autologous CD19-directed CAR T celltherapies. It may occur at the time of CRS, during the resolution ofCRS, or following resolution of CRS, and its pathophysiology is unclear.The recent ASBMT consensus further defined neurotoxicity associated withCRS as immune effector cell-associated neurotoxicity syndrome (ICANS), adisorder characterized by a pathologic process involving the CNSfollowing any immune therapy that results in activation or engagement ofendogenous or infused T cells and/or other immune effector cells (Lee etal., (2018) Biol Blood Marrow Transplant). Signs and symptoms can beprogressive and may include aphasia, altered level of consciousness,impairment of cognitive skills, motor weakness, seizures, and cerebraledema. ICANS grading was developed based on CAR Tcell-therapy-associated TOXicity (CARTOX) working group criteria usedpreviously in autologous CAR T cell trials (Neelapu et al., (2018) NEngl J Med, 377, 2531-2544). ICANS incorporates assessment of level ofconsciousness, presence/absence of seizures, motor findings,presence/absence of cerebral edema, and overall assessment of neurologicdomains by using a modified assessment tool called the ICE (immuneeffector cell-associated encephalopathy) assessment tool (see Table 21).

Evaluation of any new onset neurotoxicity should include a neurologicalexamination (including ICE assessment tool, Table 22), brain MRI, andexamination of the CSF (via lumbar puncture) as clinically indicated. Ifa brain MRI is not possible, all subjects should receive a non-contrastCT to rule out intracerebral hemorrhage. Electroencephalogram shouldalso be considered as clinically indicated. Endotracheal intubation maybe needed for airway protection in severe cases.

Non-sedating, anti-seizure prophylaxis (e.g., levetiracetam) should beconsidered in all subjects for at least 21 days following CTX110infusion or upon resolution of neurological symptoms (unless theinvestigator considers the antiseizure medication to be contributing tothe detrimental symptoms). Subjects who experience ICANS grade ≥2 shouldbe monitored with continuous cardiac telemetry and pulse oximetry. Forsevere or life-threatening neurologic toxicities, intensive caresupportive therapy should be provided. Neurology consultation shouldalways be considered. Monitor platelets and for signs of coagulopathy,and transfuse blood products appropriately to diminish risk ofintracerebral hemorrhage. Table 21 provides neurotoxicity grading andTable 23 provides management guidance.

For subjects who receive active steroid management for more than 3 days,antifungal and antiviral prophylaxis is recommended to mitigate a riskof severe infection with prolonged steroid use. Consideration forantimicrobial prophylaxis should also be given.

TABLE 21 ICANS Grading. Neuro- toxicity Grade Grade Grade Grade Domain 12 3 4 ICE 7-9 3-6 0-2 0 (subject is score ¹ unarousable and unable toundergo ICE assessment) Depressed Awakens Awakens Awakens only toSubject is level of spon- to voice tactile stimulus unarousable orconscious- taneously requires vigorous ness ² or repetitive tactilestimuli to arise; stupor or coma Seizure N/A N/A Any clinicalLife-threatening seizure, focal or prolonged seizure generalized, that(>5 min) or resolves rapidly, repetitive or nonconvulsive clinical orelectrical seizures on seizures without EEG that resolve return tobaseline with intervention in between Motor N/A N/A N/A Deep focal motorfindings ³ weakness such as hemiparesis or paraparesis Elevated N/A N/AFocal/local Diffuse cerebral ICP/ edema on edema on cerebralneuroimaging ⁴ neuroimaging, edema decerebrate or decorticate posturing,cranial nerve VI palsy, papilladema, or Cushing's triad CTCAE: CommonTerminology Criteria for Adverse Events; EEG: electroencephalogram;ICANS: immune effector cell-associated neurotoxicity syndrome; ICE:immune effector cell-associated encephalopathy (assessment tool); ICP:intracranial pressure; N/A: not applicable. ICANS grade is determined bythe most severe event (ICE score, level of consciousness, seizure, motorfindings, raised ICP/cerebral edema) not attributable to any othercause. ¹ A subject with an ICE score of 0 may be classified as grade 3ICANS if awake with global aphasia, but a subject with an ICE score of 0may be classified as grade 4 ICANS if unarousable. ² Depressed level ofconsciousness should be attributable to no other cause (e.g., sedatingmedication). ³ Tremors and myoclonus associated with immune effectortherapies should be graded according to CTCAE v5.0 but do not influenceICANS grading.

TABLE 22 ICE Assessment. Maximum Domain Assessment Score OrientationOrientation to year, month, city, hospital 4 points Naming Name 3objects (e.g., point to clock, pen, 3 points button) Following Abilityto follow commands (e.g., “Show 1 point command me 2 fingers” or “Closeyour eyes and stick out your tongue”) Writing Ability to write astandard sentence 1 point (includes a noun and verb) Attention Abilityto count backward from 100 by 10 1 point ICE score will be reported asthe total number of points (0-10) across all assessments.

The ICE assessment will be performed at screening, before administrationof CTX110 on Day 1, and on Days 2, 3, 5, 8, and 28. If a subjectexperiences CNS symptoms, the ICE assessment should continue to beperformed approximately every 2 days until resolution of symptoms. Tominimize variability, whenever possible the assessment should beperformed by the same research staff member who is familiar with ortrained in administration of the ICE assessment.

TABLE 23 ICANS Management Guidance. Severity Management Grade 2 Consideradministering dexamethasone 10 mg IV every 6 hours (or equivalentmethylprednisolone) unless subject already on equivalent dose ofsteroids for CRS. Continue dexamethasone use until event is grade Grade3 Administer dexamethasone 10 mg IV every 6 hours, unless subjectalready on equivalent dose of steroids for CRS. Continue dexamethasoneuse until event is grade Grade 4 Administer methylprednisolone 1000 mgIV per day for 3 days; if improves, manage as above. CRS: cytokinerelease syndrome; ICANS: immune effector cell-associated neurotoxicitysyndrome; IV: intravenously.

Headache, which may occur in a setting of fever or after chemotherapy,is a nonspecific symptom. Headache alone may not necessarily be amanifestation of ICANS and further evaluation should be performed.Weakness or balance problem resulting from deconditioning and muscleloss are excluded from definition of ICANS. Similarly, is intracranialhemorrhage with or without associated edema may occur due tocoagulopathies in these subjects and are also excluded from definitionof ICANS. These and other neurotoxicities should be captured inaccordance with CTCAE v5.0.

B Cell Aplasia

B cell aplasia may occur and will be monitored by followingimmunoglobulin G blood levels. IV gammaglobulin will be administered forclinically significant hypogammaglobulinemia (systemic infections)according to institutional standard of care.

Hemophagocytic Lymphohistiocytosis (HLH)

HLH has been reported after treatment with autologous CD19-directed CART cells (Barrett et al., (2014) Curr Opin Pediatr, 26, 43-49; Maude etal., (2014) N Engl J Med, 371, 1507-1517; Maude et al., (2015) Blood,125, 4017-4023; Porter et al., (2015) Sci Transl Med, 7, 303ra139;Teachey et al., (2013) Blood, 121, 5154-5157. HLH is a clinical syndromethat is a result of an inflammatory response following infusion of CAR Tcells in which cytokine production from activated T cells leads toexcessive macrophage activation. Signs and symptoms of HLH may includefevers, cytopenias, hepatosplenomegaly, hepatic dysfunction withhyperbilirubinemia, coagulopathy with significantly decreasedfibrinogen, and marked elevations in ferritin and C-reactive protein(CRP). Neurologic findings have also been observed (Jordan et al.,(2011) Blood, 118, 4041-4052; La Rosde, (2015) Hematology Am Soc HematolEduc Program, 190-196.

CRS and HLH may possess similar clinical syndromes with overlappingclinical features and pathophysiology. HLH will likely occur at the timeof CRS or as CRS is resolving. HLH should be considered if there areunexplained elevated liver function tests or cytopenias with or withoutother evidence of CRS. Monitoring of CRP and ferritin may assist withdiagnosis and define the clinical course.

If HLH is suspected:

-   -   Frequently monitor coagulation parameters, including fibrinogen.        These tests may be done more frequently than indicated in the        schedule of assessments, and frequency should be driven based on        laboratory findings.    -   Fibrinogen should be maintained ≥100 mg/dL to decrease risk of        bleeding.    -   Coagulopathy should be corrected with blood products.    -   Given the overlap with CRS, subjects should also be managed per        CRS treatment guidance in Table 19.

Cytopenias

Grade 3 neutropenia and thrombocytopenia, at times lasting more than 28days post-infusion, have been reported in subjects treated withautologous CD19-directed CAR T cell products (Kymriah USPI, 2017;Yescarta USPI, 2017). Therefore, subjects receiving CTX110 should bemonitored for such toxicities and appropriately supported. Considerationshould be given to antimicrobial and antifungal prophylaxis for anysubject with prolonged neutropenia.

G-CSF may be considered in cases of grade 4 neutropenia 21 dayspost-CTX110 infusion, when the risk of CRS has passed.

Graft Versus Host Disease

GvHD is seen in the setting of allogeneic HSCT and is the result ofimmunocompetent donor T cells (the graft) recognizing the recipient (thehost) as foreign. The subsequent immune response activates donor T cellsto attack the recipient to eliminate foreign antigen-bearing cells. GvHDis divided into acute, chronic, and overlap syndromes based on both thetime from allogeneic HSCT and clinical manifestations. Signs of acuteGvHD may include a maculopapular rash; hyperbilirubinemia with jaundicedue to damage to the small bile ducts, leading to cholestasis; nausea,vomiting, and anorexia; and watery or bloody diarrhea and crampingabdominal pain (Zeiser and Blazar, (2017) N Engl J Med, 377, 2167-2179.

To support the proposed clinical study, a nonclinical Good LaboratoryPractice (GLP)-compliant GvHD and tolerability study was performed inimmunocompromised mice at 2 doses that exceed all proposed clinical doselevels by at least 10-fold. Further, due to the specificity of CARinsertion at the TRAC locus, it is highly unlikely for a T cell to beboth CAR+ and TCR+. Remaining TCR+ cells are removed during themanufacturing process by immunoaffinity chromatography on an anti-TCRantibody column to achieve <0.5% TCR+ cells in the final product. A doselimit of 7×10⁴ TCR+ cells/kg will be imposed for all dose levels. Thislimit is lower than the limit of 1×10⁵ TCR+ cells/kg based on publishedreports on the number of allogeneic cells capable of causing severe GvHDduring SCT with haploidentical donors (Bertaina et al., (2014) Blood,124, 822-826. Through this specific editing, purification, and strictproduct release criteria, the risk of GvHD following CTX110 should below, although the true incidence is unknown. Subjects should bemonitored closely for signs of acute GvHD following infusion of CTX110.The timing of potential symptoms is unknown. However, given that CAR Tcell expansion is antigen-driven and will likely occur only in TCR−cells, it is unlikely that the number of TCR+ cells will appreciablyincrease above the number infused.

Diagnosis and grading of GvHD should be based on published criteria(Harris et al., (2016) Biol Blood Marrow Transplant, 22, 4-10), asoutlined in Table 24.

TABLE 24 Criteria for Grading Acute GvHD Skin Liver Lower GI (active(bilirubin Upper (stool Stage erythema only) mg/dL) GI output/day) 0 Noactive <2 No or <500 ml/day or (erythematous) intermittent <3episodes/day GvHD rash nausea, vomiting, or anorexia 1 Maculopapular 2-3Persistent 500-999 ml/day or rash <25% nausea, 3-4 episodes/day BSAvomiting, or anorexia 2 Maculopapular 3.1-6   1000-1500 ml/day or rash25-50% 5-7 episodes/day BSA 3 Maculopapular 6.1-15  >1500 ml/day orrash >50% >7 episodes/day BSA 4 Generalized >15 Severe abdominalerythroderma pain with or (>50% BSA) without ileus, or plus bullousgrossly bloody formation and stool (regardless desquamation of stool >5%BSA volume) BSA: body surface area; GI: gastrointestinal; GvHD: graftversus host disease.

Overall GvHD grade will be determined based on most severe target organinvolvement.

-   -   Grade 0: No stage 1-4 of any organ.    -   Grade 1: Stage 1-2 skin without liver, upper GI, or lower GI        involvement.    -   Grade 2: Stage 3 rash and/or stage 1 liver and/or stage 1 upper        GI and/or stage 1 lower GI.    -   Grade 3: Stage 2-3 liver and/or stage 2-3 lower GI, with stage        0-3 skin and/or stage 0-1 upper GI.    -   Grade 4: Stage 4 skin, liver, or lower GI involvement, with        stage 0-1 upper GI.

Potential confounding factors that may mimic GvHD such as infections andreactions to medications should be ruled out. Skin and/or GI biopsyshould be obtained for confirmation before or soon after treatment hasbeen initiated. In instance of liver involvement, liver biopsy should beattempted if clinically feasible. Sample(s) of all biopsies will also besent to a central laboratory for pathology assessment. Details of samplepreparation and shipment are contained in the Laboratory Manual.

Recommendations for management of acute GvHD are outlined in Table 25.To allow for intersubject comparability at the end of the trial,investigators should follow these recommendations except in specificclinical scenarios in which following them could put the subject atrisk.

TABLE 25 Acute GvHD Management Grade Management 1 Skin: Topical steroidsor immunosuppressants; if stage 2: prednisone 1 mg/kg (or equivalentdose). 2-4 Initiate prednisone 2 mg/kg daily (or equivalent dose). IVform of steroid such as methylprednisolone should be considered if thereare concerns with malabsorption. Steroid taper may begin afterimprovement is seen after ≥3 days of steroids. Taper should be 50%decrease of total daily steroid dose every 5 days. GI: In addition tosteroids, start anti-diarrheal agents per standard practice. GI:gastrointestinal; IV: intravenous.

Decisions to initiate second-line therapy should be made sooner forsubjects with more severe GvHD. For example, secondary therapy may beindicated after 3 days with progressive manifestations of GvHD, after 1week with persistent grade 3 GvHD, or after 2 weeks with persistentgrade 2 GvHD. Second-line systemic therapy may be indicated earlier insubjects who cannot tolerate high-dose glucocorticoid treatment (Martinet al., (2012) Biol Blood Marrow Transplant, 18, 1150-1163). Choice ofsecondary therapy and when to initiate will be based on the treatinginvestigator's clinical judgement and local practice.

Management of refractory acute GvHD or chronic GvHD will be perinstitutional guidelines. Anti-infective prophylaxis measures should beinstituted per local guidelines when treating subjects withimmunosuppressive agents (including steroids).

Hypotension and Renal Insufficiency

Hypotension and renal insufficiency have been reported with CAR T celltherapy and should be treated with IV administration of normal salineboluses according to institutional practice guidelines. Dialysis shouldbe considered when appropriate.

Study Eligibility Inclusion Criteria

To be considered eligible to participate in this study, a subject mustmeet the inclusion criteria listed below (unless indicated as optional):

1. ≥18 years of age and weight >50 kg (optional).

2. Able to understand and comply with protocol-required study proceduresand voluntarily sign a written informed consent document.

3. Diagnosed with 1 of the following B cell malignancies: Histologicallyconfirmed B cell NHLs: DLBCL NOS, high grade B cell lymphoma with MYCand BCL2 and/or BCL6 rearrangements, transformed FL, or grade 3b FL.

-   -   Confirmation of tumor histology from local pathology lab        (archival tissue from last relapse/progression [within 3 months        of enrollment] or biopsy during screening).    -   At least 1 measurable lesion that is fluorodeoxyglucose positron        emission tomography (PET)-positive, as defined by Lugano        criteria (score of 4 or 5 on Lugano criteria 5-point scale).        Previously irradiated lesions will be considered measurable only        if progression is documented following completion of radiation        therapy.

4. Refractory or relapsed disease, as evidenced by the followingcohort-specific criteria:

Two or more lines of prior therapy, including an anti-CD20 monoclonalantibody and an anthracycline-containing regimen, and have failed priorautologous hematopoietic stem cell transplantation (HSCT) or ineligiblefor or refused prior autologous HSCT. Subjects who have receivedautologous HSCT must have recovered from HSCT-related toxicities.

-   -   For refractory disease, subjects must have progressive disease        on last therapy, or have stable disease following at least 2        cycles of therapy with duration of stable disease of up to 6        months.    -   For subjects with transformed FL, subjects must have received at        least 1 line of chemotherapy for disease after transformation to        DLBCL.

5. Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1.

6. Meets criteria to undergo LD chemotherapy and CAR T cell infusion.

7. Adequate organ function:

-   -   Renal: Estimated glomerular filtration rate >50 mL/min/1.73 m².    -   Liver: Aspartate transaminase or alanine transaminase <3× upper        limit of normal (ULN); total bilirubin <1.5×ULN (for subjects        with Gilbert's syndrome, total bilirubin <2 mg/dL).    -   Cardiac: Hemodynamically stable and left ventricle ejection        fraction ≥45% by echocardiogram.    -   Pulmonary: Oxygen saturation level on room air >91% per pulse        oximetry.

8. Female subjects of childbearing potential (postmenarcheal with anintact uterus and at least 1 ovary, who are less than 1 yearpostmenopausal) must agree to use acceptable method(s) of contraceptionfrom enrollment through at least 12 months after CTX110 infusion.

9. Male subjects must agree to use effective contraception fromenrollment through at least 12 months after CTX110 infusion.

10. Agree to participate in an additional long-term follow-up studyafter completion of this study.

Exclusion Criteria

To be eligible for entry into the study, the subject must not meet anyof the exclusion criteria listed below:

1. Eligible for and agrees to autologous HSCT.

2. Treatment with the following therapies as described below:

-   -   Prior treatment with any gene therapy or genetically modified        cell therapy, including CAR T cells.    -   Prior treatment with a CD19-directed antibody, bispecific T cell        engager, or antibody-drug conjugate, unless there is confirmed        CD19 expression (by immunohistochemistry or flow cytometry)        after progression or relapse following most recent CD19-directed        treatment.

3. Prior allogeneic HSCT.

4. Known contraindication to cyclophosphamide, fludarabine, or any ofthe excipients of CTX110 product.

5. Detectable malignant cells from cerebrospinal fluid (CSF) or magneticresonance imaging (MRI) indicating brain metastases during screening, ora history of central nervous system (CNS) involvement by malignancy (CSFor imaging).

6. History of a seizure disorder, cerebrovascular ischemia/hemorrhage,dementia, cerebellar disease, or any autoimmune disease with CNSinvolvement.

7. Unstable angina, clinically significant arrhythmia, or myocardialinfarction within 6 months prior to screening.

8. Uncontrolled, acute life-threatening bacterial, viral, or fungalinfection.

9. Positive for presence of human immunodeficiency virus (HIV) type 1 or2, or active hepatitis B virus (HBV) or hepatitis C virus (HCV)infection. Subjects with prior history of HBV or HBC infection who havedocumented undetectable viral load (by quantitative polymerase chainreaction [PCR] or nucleic acid testing) are permitted. Infectiousdisease testing (HIV-1, HIV-2, HCV antibody and PCR, HBV surfaceantigen, HBV surface antibody, HBV core antibody) performed within 30days of signing the informed consent form may be considered for subjecteligibility.

10. Previous or concurrent malignancy, except basal cell or squamouscell skin carcinoma, adequately resected and in situ carcinoma ofcervix, or a previous malignancy that was completely resected and hasbeen in remission for ≥5 years.

11. Radiation therapy within 14 days of enrollment.

12. Use of systemic antitumor therapy or investigational agent within 14days or 5 half-lives, whichever is longer, of enrollment. Exceptions aremade for 1) prior inhibitory/stimulatory immune checkpoint moleculetherapy, which is prohibited within 3 half-lives of enrollment, and 2)rituximab use within 30 days prior to screening is prohibited.

13. Primary immunodeficiency disorder or active autoimmune diseaserequiring steroids and/or other immunosuppressive therapy.

14. Diagnosis of significant psychiatric disorder or other medicalcondition that could impede the subject's ability to participate in thestudy.

15. Women who are pregnant or breastfeeding.

Statistical Methods Sample Size

The sample size in the dose escalation part of the study will beapproximately 6 to 54 subjects, depending on the number of dose levelsand cohorts evaluated, and the occurrence of DLTs.

If the study proceeds to cohort expansion, an optimal Simon 2-stagedesign will be employed. The sample size for each cohort will depend onthe assumption of effect size for the specific indication.

For expansion of Cohort A, in the first stage, up to 30 subjects will beenrolled. If 10 or more of the first 30 subjects in the full analysisset achieve an objective response, the study will expand enrollment toinclude an additional 47 subjects (77 total) in the second stage. Afinal sample size of 77 subjects will have 90% power (α=0.05, 2-sidedtest) to test for a difference between a ORR of 45% with CTX110 and anORR of 26%, the estimated ORR to standard salvage therapy in patientswith relapsed/refractory DLBCL.

As in Cohort A, upon completion of the dose escalation part of thestudy, Cohort B may go on to cohort expansion after a protocolamendment.

To date, all subjects that participated in this study have completedStage 1 (eligibility screening) within 14 days. One subject completedStage 1 within 2 days. A subject who met the eligibility criteriastarted lymphodepleting therapy within 24 hours of completing Stage 1.All eligible subjects have completed the screening period (stage 1) andreceived LD chemotherapy in less than 15 days, with one patientcompleting screening and starting an LD chemo dose within 72 hrs. Someof the eligible subjects have DLBCL (e.g., NOS, high grade); others havetransformed FL and Richter's transformation.

All subjects receiving LD chemotherapy have progressed to receiving theDL1 or DL2 dose of CTX110 within 2-7 days following completion of the LDchemotherapy. Results obtained from these patients to date aresummarized below.

Subjects in both DL1 and DL2 doses experienced decreased tumor metabolicactivity (FDG uptake on PET scan) and/or decrease in tumor size. A dosedependent response has been observed, including a complete and durableresponse for >60 days at DL2. None of the treated patients exhibited anyDLTs so far. Further, the allogeneic CAR-T cell therapy exhibiteddesired pharmacokinetic features in the treated human subjects,including CAR-T cell expansion and persistence after infusion. A dosedependent effect has also been observed in both CTX110 expansion andpersistence. All subjects in DL2 have exhibited CTX110 expansion andpersistence Up to 90-fold expansion of CTX110 in peripheral blood hasbeen observed in one subject. Further, persistence of CTX110 cells canbe detected in DL2 subjects at least 8-10 days following treatment andhas been detected up to 28 days post-infusion.

SEQUENCE TABLE SEQ ID NO Description Sequence 1 TRAC gene-editAAGAGCAACAAATCTGACT 2 TRAC gene-editAAGAGCAACAGTGCTGTGCCTGGAGCAACAAATCTGACT AAGAGCAACAAATCTGACT 3TRAC gene-edit AAGAGCAACAGTGCTGGAGCAACAAATCTGACT AAGAGCAACAAATCTGACT 4TRAC gene-edit AAGAGCAACAGTGCCTGGAGCAACAAATCTGACT AAGAGCAACAAATCTGACT 5TRAC gene-edit AAGAGCAACAGTGCTGACTAAGAGCAACAAATCTGACT 6 TRAC gene-editAAGAGCAACAGTGCTGTGGGCCTGGAGCAACAAATCTGA CT AAGAGCAACAAATCTGACT 7TRAC gene-edit AAGAGCAACAGTGCTGGCCTGGAGCAACAAATCTGACTAAGAGCAACAAATCTGACT 8 TRAC gene-editAAGAGCAACAGTGCTGTGTGCCTGGAGCAACAAATCTGA CT AAGAGCAACAAATCTGACT 9B2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 10 B2M gene-editCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 11 B2M gene-editCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 12 B2M gene-editCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGATAGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCC GCT 13 B2M gene-editCGTGGCCTTAGCTGTGCTCGCGCTATCCAGCGTGAGTCTC TCCTACCCTCCCGCT 14B2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGTGGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGC T 15 sgRNAnnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu 16 sgRNAnnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc 17 sgRNAn₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcu₍₁₋₈₎ 18 TRAC sgRNA (TA-1)AGAGCAACAGUGCUGUGGCCguuuuagagcuagaaauagcaaguuaa unmodifiedaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcUUUU 19TRAC sgRNA spacer AGAGCAACAGUGCUGUGGCC unmodified 20 B2M sgRNAGCUACUCUCUCUUUCUGGCCguuuuagagcuagaaauagcaaguuaaa unmodifiedauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcUUUU 21 B2M sgRNA spacerGCUACUCUCUCUUUCUGGCC unmodified 22 TRAC sgRNA (TA-1)A*G*A*GCAACAGUGCUGUGGCCguuuuagagcuagaaauagcaagu modifieduaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U* *: 2′-O-methylU*U phosphorothioate residue 23 TRAC sgRNA spacerA*G*A*GCAACAGUGCUGUGGCC modified *: 2′-O-methyl phosphorothioate residue24 B2M sgRNA G*C*U*ACUCUCUCUUUCUGGCCguuuuagagcuagaaauagcaagu modifieduaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U* *: 2′-O-methylU*U phosphorothioate residue 25 B2M sgRNA spacer G*C*U*ACUCUCUCUUUCUGGCCmodified *: 2′-O-methyl phosphorothioate residue 26 TRAC targetAGAGCAACAGTGCTGTGGCC sequence 27 B2M target sequenceGCTACTCTCTCTTTCTGGCC 28 TRAC target AGAGCAACAGTGCTGTGGCC (TGG)sequence with (PAM) 29 B2M target sequence GCTACTCTCTCTTTCTGGCC (TGG)with (PAM) 30 signal peptide MLLLVTSLLLCELPHPAFLLIP 31 signal peptideMALPVTALLLPLALLLHAARP 32 CD8a transmembrane IYIWAPLAGTCGVLLLSLVITLYdomain 33 4-1BB nucleotide AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAAsequence CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA TGTGAACTG 34 4-1BB amino acidKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL sequence 35 CD28 nucleotideTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATA sequenceTGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTC C 36 CD28 amino acidSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS sequence 37 CD3-zeta nucleotideCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATAT sequenceCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACT GCATATGCAGGCCCTGCCTCCCAGA 38CD3-zeta amino acid RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG sequenceRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 39 FMC63-28ZATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCC (FMC63-CD8[tm]-TCATCCAGCGTTCTTGCTGATCCCCGATATTCAGATGACT CD28[co-stimulatoryCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCGA domain]-CD3z)GTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATAT GCAGGCCCTGCCTCCCAGA 40 FMC63-28ZMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTI (FMC63-CD8[tm]-SCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS CD28[co-stimulatoryGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEI domain]-CD3z)TGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCT Amino AcidVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR 41 TRAC-LHA (800 bp)GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGG ACTTCA 42 TRAC-RHA (800 bp)TGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGC TCAATGAGAAAGG 43 EF1aGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCA AAGTTTTTTTCTTCCATTTCAGGTGTCGTGA44 GM-CSF signal ATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCC peptideTCATCCAGCGTTCTTGCTGATCCCC 45 GM-CSF signal MLLLVTSLLLCELPHPAFLLIPpeptide 46 Anti-CD19 scFv GATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGC AGGGGACTTCTGTCACAGTCAGTAGT 47CD19 scFv amino DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD acid sequenceGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA Linker underlinedTYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 48 CD8a extracellular +GCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGA CD8a transmembrane +CCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCAC 5′ LinkerCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGC (underlined)CGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTG TATTGTAATCACAGGAATCGC 49CD8a extracellular + TTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCD8a transmembrane CCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTC(without linker) TCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATC ACAGGAATCGC 50CD8a extracellular + FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGCD8a transmembrane AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR 51 CD19 VHEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 52 CD19 VLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA TYFCQQGNTLPYTFGGGTKLEIT 53CD19 linker GSTSGSGKPGSGEGSTKG 54 LHA to RHAGAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCCAGCGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGA GAAAGG 55 spCas9MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFD SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 56 rAAVCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCCAGCGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGGTAACCACGTGCGGACCGAGGCTGCAGCGTCGTCCTCCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG AGCGCGCAGCTGCCTGCAGG *indicatesa nucleotide with a 2′-O-methyl phosphorothioate modification.“n” refers to the spacer sequence at the 5′ end.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

1. A method for treating a B-cell malignancy in a human patient, themethod comprising: (i) subjecting a human patient having a B-cellmalignancy to a lymphodepletion treatment; and (ii) administering to thehuman patient a population of genetically engineered T cells after step(i), wherein the population of genetically engineered T cells comprisingT cells that comprise: (a) a disrupted T cell receptor alpha constant(TRAC) gene, (b) a nucleic acid coding for a chimeric antigen receptor(CAR) that binds CD19, wherein the CAR comprises an anti-CD19 singlechain variable fragment (scFv) that comprises a heavy chain variableregion set forth in SEQ ID NO: 51, and a light chain variable region setforth in SEQ ID NO: 52, and wherein the nucleic acid is inserted in thedisrupted TRAC gene, and (c) a disrupted beta 2-microglobulin (β2M)gene; wherein the population of genetically engineered T cells isadministered to the human patient at a dose of about 1×10⁷ to about1×10⁹ CAR⁺ T cells.
 2. The method of claim 1, wherein the disrupted TRACgene comprises a deletion of a fragment comprising the nucleotidesequence of SEQ ID NO: 26,
 3. The method of claim 1, wherein thepopulation of genetically engineered T cells administered to the humanpatient per dose contains no more than 7×10⁴ TCR⁺ T cells/kg.
 4. Themethod of claim 1, wherein the lymphodepletion treatment in step (i)comprises co-administration to the human patient fludarabine at about 30mg/m² and cyclophosphamide at about 500-750 mg/m² per day for threedays.
 5. The method of claim 4, wherein the lymphodepletion treatment instep (i) comprises co-administration to the human patient fludarabine atabout 30 mg/m² and cyclophosphamide at about 500 mg/m² per day for threedays, or fludarabine at about 30 mg/m² and cyclophosphamide at about 750mg/m² per day for three days.
 6. The method of claim 1, wherein thepopulation of genetically engineered T cells is administered to thehuman patient at a dose of about 1×10⁷, about 3×10⁷, about 1×10⁸, about3×10⁸, or about 1×10⁹ CAR′ T cells.
 7. The method of claim 1, whereinprior to step (i), the human patient does not show one or more of thefollowing features: (a) significant worsening of clinical status, (b)requirement for supplemental oxygen to maintain a saturation level ofgreater than 91%, (c) uncontrolled cardiac arrhythmia, (d) hypotensionrequiring vasopressor support, (e) active infection, and (f) grade ≥2acute neurological toxicity.
 8. The method of claim 1, wherein step (i)is performed about 2-7 days prior to step (ii).
 9. The method of claim1, wherein after step (i) and prior to step (ii), the human patient doesnot show one or more of the following features: (a) active uncontrolledinfection; (b) worsening of clinical status compared to the clinicalstatus prior to step (i); and (c) grade ≥2 acute neurological toxicity.10. The method of claim 1, further comprising (iii) monitoring the humanpatient for development of acute toxicity after step (ii); and (iv)managing the acute toxicity if occurs.
 11. The method of claim 10,wherein step (iii) is performed for at least 28 days afteradministration of the population of genetically engineered T cells. 12.The method of claim 10, wherein the acute toxicity comprises tumor lysissyndrome (TLS), cytokine release syndrome (CRS), immune effectorcell-associated neurotoxicity syndrome (ICANS), B cell aplasia,hemophagocytic lymphohistiocytosis (HLH), cytopenia, graft-versus-hostdisease (GvHD), hypertension, renal insufficiency, or a combinationthereof.
 13. The method of claim 1, wherein the B cell malignancy isnon-Hodgkin lymphoma, which optionally is selected from the groupconsisting of diffuse large B cell lymphoma (DLBCL), high grade B celllymphoma with MYC and BCL2 and/or BCL6 rearrangement, transformedfollicular lymphoma (FL), and grade 3b FL.
 14. The method of claim 13,wherein DLBCL is DLBCL not otherwise specified (NOS).
 15. The method ofclaim 1, wherein the human patient has at least one measurable lesionthat is fluorodeoxyglucose positron emission tomography (PET)-positive.16. The method of claim 1, wherein the B cell malignancy is refractoryand/or relapsed.
 17. The method of claim 1, wherein the human patienthas undergone one or more lines of prior anti-cancer therapies.
 18. Themethod of claim 17, wherein the human patient has undergone two or morelines of prior anti-cancer therapies.
 19. The method of claim 17,wherein the prior anti-cancer therapies comprise an anti-CD20 antibody,an anthracycline-containing regimen, or a combination thereof.
 20. Themethod of claim 17, wherein the human patient has refractory or relapsedtransformed FL and has undergone at least one line of chemotherapy fordisease after transformation to DLBCL.
 21. The method of claim 16,wherein the B cell malignancy is refractory, and the human patient hasprogressive disease on last therapy, or has stable disease following atleast two cycles of therapy with duration of stable disease of up to 6months.
 22. The method of claim 1, wherein the human patient has failedprior autologous hematopoietic stem cell transplantation (HSCT) orineligible for prior autologous HSCT.
 23. The method of claim 1, whereinthe human patient is subject to an additional anti-cancer therapy aftertreatment with the population of genetically engineered T cells.
 24. Themethod of claim 1, wherein the human patient has one or more of thefollowing features: (a) has an Eastern Cooperative Oncology Group (ECOG)performance status 0 or 1; (b) adequate renal, liver, cardiac, and/orpulmonary function; (c) free of prior gene therapy or modified celltherapy; (d) free of prior treatment comprising an anti-CD19 antibody;(e) free of prior allogeneic HSCT; (f) free of detectable malignantcells from cerebrospinal fluid; (g) free of brain metastases; (h) freeof prior central nervous system disorders; (i) free of unstable angina,arrhythmia, and/or myocardial infarction; (j) free of uncontrolledinfection; (k) free of immunodeficiency disorders or autoimmunedisorders that require immunosuppressive therapy; and (l) free ofinfection by human immunodeficiency virus, hepatitis B virus, orhepatitis C virus.
 25. The method of claim 1, wherein the anti-CD19 scFvcomprises the amino acid sequence of SEQ ID NO:
 47. 26. The method ofclaim 25, wherein the CAR that binds CD19 comprises the amino acidsequence of SEQ ID NO:
 40. 27. The method of claim 1, wherein thenucleic acid encoding the anti-CD19 CAR is inserted at the site ofdeletion in the disrupted TRAC gene.
 28. The method of claim 1, whereinthe disrupted TRAC gene comprises the nucleotide sequence of SEQ ID NO:54.
 29. The method of claim 1, wherein the disrupted 32M gene in thepopulation of genetically engineered T cells comprises at least one ofthe nucleotide sequence set forth in SEQ ID NOs: 9-14.
 30. The method ofclaim 1, wherein the population of genetically engineered T cells isallogeneic.
 31. The method of claim 1, wherein at least 90% of the Tcells in the population of genetically engineered T cells do not expressa detectable level of TCR surface protein.
 32. The method of claim 1,wherein at least 70% of the T cells in the population of geneticallyengineered T cells do not express a detectable level of TCR surfaceprotein, wherein at least 50% of the T cells in the population ofgenetically engineered T cells do not express a detectable level of B2Msurface protein; and/or wherein at least 30% of the T cells in thepopulation of genetically engineered T cells express a detectable levelof the CAR.
 33. The method of claim 32, wherein at least 99.5% of the Tcells in the population of genetically engineered T cells do not expressa detectable level of TCR surface protein.
 34. The method of claim 1,wherein at least 70% of the T cells in the population of geneticallyengineered T cells do not express a detectable level of B2M surfaceprotein.
 35. The method of claim 34, wherein at least 85% of the T cellsin the population of the genetically engineered T cells do not express adetectable level of B2M surface protein.
 36. The method of claim 1,wherein at least 50% of the T cells in the population of geneticallyengineered T cells express a detectable level of the CAR.
 37. The methodof claim 36, wherein at least 70% of the T cells in the population ofgenetically engineered T cells express a detectable level of the CAR.38. The method of claim 1, wherein the population of geneticallyengineered T cells are administered to the human patient via intravenousinfusion.
 39. The method of claim 1, wherein the population ofgenetically engineered T cells are suspended in a cryopreservationsolution.
 40. A pharmaceutical composition for use in treating a B-cellmalignancy, the pharmaceutical composition comprising a population ofgenetically engineered T cells that comprises: (a) a disrupted T cellreceptor alpha constant (TRAC) gene, which optionally comprises adeletion of a fragment comprising the nucleotide sequence of SEQ ID NO:26, (b) a nucleic acid coding for a chimeric antigen receptor (CAR) thatbinds CD19, wherein the CAR comprises an anti-CD19 single chain variablefragment (scFv) that comprises a heavy chain variable region set forthin SEQ ID NO: 51 and a light chain variable region set forth in SEQ IDNO: 52, and wherein the nucleic acid is inserted in the disrupted TRACgene, and (c) a disrupted beta 2-microglobulin (32M) gene; wherein thecomposition comprises about 1×10⁷ to about 1×10⁹ CAR⁺ T cells. 41-42.(canceled)